PCR-positive Dogs Research Articles

Enhanced Production of IL-10 in PCR-Positive Dogs Infected with E. canis and A. phagocytophilum Facilitate Specific Immune Responses

Infection of dogs with the tick-borne rickettsiae Ehrlichia and Anaplasma provokes an immune response mediating the pathology and bacterial resistance. IL-10 is the main anti-inflammatory cytokine and plays a multifaceted role in host protection. The study aimed to investigate circulating IL-10 in 32 dogs naturally infected with A. phagocytophilum and E. canis, identified by PCR positivity, and 33 PCR-negative animals which tested positive for antibodies against these pathogens, as well as 22 healthy animals. The highest quantity of IL-10, measured by ELISA, was observed among dogs positive simultaneously for anti-E. canis and anti-A. phagocytophilum IgG antibodies, followed by dogs positive for anti-E. canis only. The concentration of IL-10 in PCR-positive dogs was almost three and a half times higher than that measured in the control group (77.09 ± 23.61 pg./mL vs. 21.55 ± 4.61 pg./mL; p = 0.0015) and five times higher than the concentration of interleukin in PCR-negative animals (77.09 ± 23.61 pg./mL vs. 14.86 ± 3.01 pg./mL; p = 0.000016). The highest level of IL-10 was observed in PCR-positive dogs with mixed infection (120.54 ± 44.18), followed by the level in PCR-positive dogs for E. canis only (78.81 ± 16.92). The lowest level of IL-10 was observed in PCR-positive dogs for A. phagocytophilum only (56.32 ± 12.68). We may suggest that infection with E. canis and A. phagocytophilum stimulates the IL-10 production in dogs, which may facilitate specific antibody responses.

Veterinary perspectives on the urbanization of leishmaniosis in Morocco

BackgroundLeishmaniosis caused by Leishmania infantum, L. major and L. tropica is endemic in Morocco. Growing evidence of both human and canine Leishmania infections in urban centres has been reported. Since many forms of the disease are zoonotic, veterinarians play an important role in leishmaniosis control by intervening at the parasite host level. This study aimed to bring together One Health principles to connect canine and feline leishmaniosis epidemiology within urban centres of Morocco (Rabat and Fez) and assess the level of awareness of Moroccan veterinarians about facing this threat.MethodsA molecular survey was conducted for Leishmania DNA detection in canine (n = 155) and feline (n = 32) whole-blood samples. Three conventional polymerase chain reaction (PCR) protocols were implemented. The first PCR aimed at identifying infected animals by targeting Leishmania spp. kinetoplast minicircle DNA (kDNA). The second and third PCR targeted the Leishmania internal transcribed spacer region (ITS-1) and the Leishmania small subunit ribosomal RNA (SSUrRNA) gene, respectively, aiming at identification of the infecting species after Sanger sequencing-positive amplicons. Total immunoglobulin G (IgG) against Leishmania spp. was evaluated in 125 dogs by enzyme-linked immunosorbent assays (ELISA) using an in-house protocol, including three Leishmania-specific antigens (SPLA, rKDDR and LicTXNPx). Sera from 25 cats were screened for total IgG to Leishmania spp. by an indirect immunofluorescence antibody test (IFAT). An online questionnaire was presented to Moroccan veterinarians addressing their knowledge and practices towards animal leishmaniosis.ResultsOverall, 19.4% of the dogs tested positive for Leishmania kDNA and ITS-1 and sequencing revealed infection with L. infantum among PCR-positive dogs. These animals presented a wide range of ELISA seropositivity results (16.7%, 34.9% and 51.6%) according to the tested antigens (rKDDR, SPLA and LicTXNPx, respectively). Use of kDNA-PCR revealed 12.5% cats positive to Leishmania spp. otherwise found to be seronegative by IFAT.ConclusionsA considerable prevalence of infection was identified in dogs from urban centres of Morocco. Additionally, this is the first report of feline infection with Leishmania spp. in this country and in urban settings. Moroccan veterinarians are aware that animal leishmaniosis is endemic in Morocco, representing a public health threat, and are knowledgeable about canine leishmaniosis diagnosis and treatment.Graphical

Domestic Dog Infection with Trypanosoma cruzi from Northern and Southern Regions of Mexico.

Background: Chagas disease or American trypanosomiasis, caused by Trypanosoma cruzi and vectored by triatomines, affects millions of people worldwide. In endemic countries including Mexico, infections in domestic animals, such as dogs, may affect the risk of human disease when they serve as a source of infection to vectors that subsequently infect humans. Materials and Methods: We conducted a cross-sectional study of 296 dogs from two cities near the northern and southern borders of Mexico: Reynosa, Tamaulipas, and Tuxtla Gutierrez, Chiapas. Infection was measured based on testing of blood using T. cruzi quantitative PCR (qPCR) and up to three antibody detection assays. The StatPak immunochromatographic assay was used to screen samples and the indirect fluorescent antibody (IFA) and multiplex microsphere immunoassay (MIA) tests were used as secondary tests on all samples that screened positive and a subset of negatives. Serologic positivity was defined based on reactivity on at least two independent tests. Results: Of the 280 samples tested for parasite DNA, two (0.7%) were positive, one of which (0.4%) was confirmed as T. cruzi discrete typing unit TcIV. Overall, 72 (24.3%) samples were reactive for T. cruzi antibodies via StatPak of which 8 were also positive using MIA and 2 were also positive using IFA (including one of the PCR-positive dogs). Overall, nine dogs (3.4%) met study criteria of positivity based on either/both serology or PCR tests. Positive dogs were found in both regions of Mexico; five (2.7%) from Reynosa and four (3.6%) from Tuxtla Gutierrez. We found no association between infection status and state of origin, sex, age group, breed group, neighborhood, and whether other pets lived in the home. Conclusion: Our results re-emphasize dogs' utility as sentinels for T. cruzi in Mexico and underscore the need for improved veterinary diagnostic tests and parasite surveillance at the household level in endemic countries.

Survival outcomes, low awareness, and the challenge of neglected leptospirosis in dogs.

Leptospirosis is a globally neglected zoonotic disease with significant morbidity and mortality in dogs, particularly in resource-limited settings. This study aimed to characterize prognostic factors and survival outcomes in dogs with suspected leptospirosis, emphasizing the potential underestimation of disease burden. This retrospective study was conducted using medical records of dogs diagnosed with urinary Leptospira polymerase chain reaction (PCR). Urinary Leptospira PCR was positive in 22 dogs and negative in 62. Azotemia was present in approximately two-thirds of both groups, with no predictive value identified between PCR-positive and PCR-negative dogs. However, PCR-positive dogs exhibited significantly shorter survival times for both all-cause mortality (median 60 days, range: 8-601 days) and leptospirosis-related death (median 27 days, range: 8-67 days) compared to PCR-negative dogs (median 402 days, range: 7-812 days) (p < 0.01). The neutrophil-to-lymphocyte ratio (NLR) in-dependently predicted leptospirosis-related death (HR = 1.073, 95%CI: 1.02-1.13, p = 0.01), while the BUN-to-creatinine ratio predicted all-cause mortality (HR = 1.02, 95% CI: 1.003-1.03, p = 0.02). Our findings underscore the severity of leptospirosis in older dogs, particularly those with azotemia or positive PCR results. NLR and BUN to creatinine ratios could be valuable tools for risk assessment and guiding treatment strategies in this vulnerable population.

The Prevalence, Seroprevalence, and Risk Factors of Tick-Borne Encephalitis Virus in Dogs in Lithuania, a Highly Endemic State.

The rising awareness and increasing number of case reports of tick-borne encephalitis (TBE) in dogs indicate that the virus might be an important tick-borne pathogen in dogs, especially in endemic areas. Therefore, the aim of the present study was to investigate the prevalence rate of TBEV RNA and TBEV-specific antibodies in clinical samples of dogs living in a highly endemic region of Lithuania and to evaluate the main risk factors for severe disease course and death. The blood samples (n = 473) of dogs were collected in two veterinary clinics in central Lithuania. Tick-borne encephalitis virus (TBEV) RNA was detected in 18.6% (88/473; CI 95% 15.2-22.4) and TBEV-specific antibodies were found in 21.6% (102/473; CI 95% 17.9-25.6) of dog blood serum samples after confirmation with a virus neutralization test. The death/euthanasia rate was 18.2% (16/88; CI 95% 10.8-27.8) in PCR-positive dogs. Male dogs were more likely to develop neurological symptoms (p = 0.008). Older dogs (p = 0.003), dogs with the presence of neurological symptoms (p = 0.003), and dogs with the presence of TBEV-specific antibodies (p = 0.024) were more likely to experience worse outcomes of the disease. The results of the present study demonstrate that TBEV is a common and clinically important pathogen in dogs in such endemic countries as Lithuania.

A blinded, randomized and controlled multicenter clinical trial to assess the efficacy and safety of Leisguard® as an immunotherapeutic treatment for healthy Leishmania infantum-seropositive dogs

BackgroundDomperidone (Leisguard®) is an immunomodulatory drug used as a preventive measure in healthy dogs. However, no studies have been published in healthy Leishmania infantum-seropositive dogs. The aim of this study was to evaluate the clinical efficacy and safety of domperidone as immunotherapy in Leishmania-seropositive healthy dogs.MethodsSixty-seven dogs were treated with domperidone at 0.5 mg/kg and 44 dogs received placebo, once daily for 4 consecutive weeks. Monthly treatments were repeated every 4 months until the end of the 1-year follow-up period. Veterinary examinations were performed on days 0, 30, 120, 150, 240, 270 and 360. Samples of blood and urine were collected on days 0, 120, 240 and 360 for routine laboratory tests and quantitative in-house ELISA for the detection of L. infantum-specific antibodies. Furthermore, Leishmania real-time PCR and IFN-γ ELISA were performed at day 0 and the end of the study. Dogs that developed disease were withdrawn from the study and classified as sick dogs. Adverse drug reactions were reported.ResultsThirty dogs developed disease during the follow-up period: 13/67 (19.4%) in the group treated with domperidone and 17/44 (38.6%) in the placebo-treated group (P = 0.03). Low-seropositive dogs treated with domperidone (4/40, 9.1%) were significantly less likely to develop disease compared to low-seropositive dogs treated with placebo (7/24, 29.2%; P = 0.04), while no differences were found between domperidone (9/23, 39.1%) and placebo (10/20, 50%) in medium- to high-seropositive dogs. At the end of the study, a higher proportion of Leishmania PCR-positive dogs was observed in the placebo-treated group (16/33, 48.5%) compared to the domperidone group (13/51, 25.5%; P = 0.04). Furthermore, low-seropositive dogs treated with domperidone with an increase of IFN-γ concentration presented a higher increase than those treated with placebo at the end of the study. Four dogs treated with domperidone presented self-limiting diarrhea.ConclusionsHealthy dogs with low L. infantum antibody levels treated with domperidone were less likely to develop disease compared to placebo-treated dogs. Furthermore, domperidone presented a good safety profile.Graphical abstract

Dirofilaria immitis in pet dogs from the metropolitan area of the Colombian Caribbean

A cross-sectional study was carried out to determine the frequency and factors associated with Dirofilaria immitis infection in pet dogs in the metropolitan area of the Colombian Caribbean (northern Colombia). A total of 173 dogs were analyzed by a commercial rapid immunochromatographic test (RIT) and a nested PCR of the cytochrome oxidase subunit I gene, in parallel. Ninety-two (53.2%) of the dogs showed positive results to the RIT, while 59 (34.1%) animals had D. immitis DNA by PCR. Positivity to one or both diagnostic techniques was detected in 104 (60.1%; CI95%: 53.8–67.4) of the sampled dogs. In PCR-positive dogs, phylogenetic analyses evidenced high nucleotide identity (100%) with sequences previously obtained from mosquitoes, dogs and other mammals in different countries. Exercise intolerance (p = 0.002; OR: 2.33; CI95%: 1.37–3.96) and thrombocytopenia (p = < 0.001; OR: 1.95; CI95%: 1.11–3.43) were the main factors associated with D. immitis infection in dogs. The high frequency of D. immitis in dogs indicates a wide distribution of this parasite in the metropolitan area of the Colombian Caribbean, which can be of animal and public health concern. Our results highlight the need to combine different methods to increase the diagnostic accuracy of D. immitis.

A second-generation, point-of-care immunoassay provided improved detection of Anaplasma and Ehrlichia antibodies in PCR-positive dogs naturally infected with Anaplasma or Ehrlichia species.

A validated second-generation SNAP 4Dx Plus (Idexx) incorporates new peptides for improved detection of antibodies against Anaplasma and Ehrlichia tick-borne pathogens in dogs. We compared the first- and second-generation SNAP 4Dx Plus using dogs naturally infected with Anaplasma or Ehrlichia species, or dogs seroreactive by an E. canis indirect fluorescent antibody test (IFAT). The second-generation immunoassay was more sensitive than the first-generation for dogs infected with A. phagocytophilum (51.1% and 29.2%, respectively), A. platys (63.6% and 35.3%, respectively), E. canis (96.2% and 88.3%, respectively), or E. ewingii (73.7% and 70.8%, respectively), and for dogs seroreactive by E. canis IFAT (87.3% and 83.9%, respectively). The second-generation immunoassay detected significantly more Anaplasma- or Ehrlichia-infected dogs that were Anaplasma (p < 0.001) or Ehrlichia (p = 0.031) seroreactive, respectively, than did the first-generation test. When Ehrlichia seroreactivity by E. canis IFAT and both immunoassays was compared, significantly more E. canis-infected dogs were seroreactive by E. canis IFAT than the first-generation (p = 0.006) but not the second-generation (p = 0.125) immunoassay. Significantly more E. ewingii-infected dogs were seroreactive by the first- (p = 0.011) and second-generation (p = 0.049) immunoassays than the E. canis IFAT. Medical records available for 7 dogs that were Anaplasma seroreactive by the second-generation but not the first-generation immunoassay revealed case management decisions that might have been different with an immediate anaplasmosis diagnosis, including earlier doxycycline therapy and less hospitalization. The second-generation SNAP 4Dx Plus test offered improved serologic detection of Anaplasma and Ehrlichia in naturally infected dogs.

Domestic dogs carriers of Leishmania infantum, Leishmania tropica and Crithidia fasciculata as potential reservoirs for human visceral leishmaniasis in northeastern Iran.

In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%-80% of seropositive dogs are asymptomatic for Leishmania infection. The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran. Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti-Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR. Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody-positive dogs and 19 of the 25 (76%) PCR-positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR-RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR-positive samples. The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs.

Helicobacter Species and Their Association with Gastric Pathology in a Cohort of Dogs with Chronic Gastrointestinal Signs.

Simple SummaryHelicobacter spp. represent spiral-shaped Gram-negative bacteria that can live in the acidic environment of the stomach. While their association with peptic ulcers and gastric neoplasia in people has been clearly documented, their pathogenic potential in dogs is less well defined. Helicobacter pylori, the most prevalent Helicobacter infecting people, does not seem to represent a significant problem in small animals. The aims of this study were to determine the prevalence of individual gastric Helicobacter species in dogs with chronic gastrointestinal signs, their association with gastric lesions, and to compare diagnostic techniques used to detect Helicobacter infection in dogs.Prevalence of individual Helicobacter species, data evaluating their association with gastric pathology and comparison of accuracy of diagnostic techniques are limited. The aims of this study were to determine the prevalence of gastric Helicobacter species, their association with gastric pathology, and to compare diagnostic techniques. Gastric biopsies from 84 privately-owned dogs with chronic gastrointestinal signs were obtained endoscopically. Helicobacters were detected using PCR, cytology, urease test, and histopathology. PCR detected helicobacters in 71.4% of dogs. Helicobacter heilmannii sensu stricto (s.s.) was the predominant species. Mixed infection was detected in 40% of PCR positive dogs. Gastritis was diagnosed in 38.5% of Helicobacter positive and 47.4% of Helicobacter negative dogs. Mono-infection was associated with 2.4 times increased odds of having more severe inflammation compared to mixed infection. Erosions and ulcers were common endoscopic lesions. Cytology had sensitivity/specificity of 88.3/91.7%. Association between infection and lymphoid follicular hyperplasia was demonstrated.

Infection and seroprevalence of Borrelia persica in domestic cats and dogs in Israel

BackgroundRelapsing fever borreliosis is an infectious disease caused by bacteria of the genus Borrelia, inflicting recurrent episodes of fever and spirochetemia in humans. Borrelia persica, the causative agent of relapsing fever in Israel, is prevalent over a broad geographic area that extends from India to Egypt. It is transmitted by the soft tick Ornithodoros tholozani and causes disease in humans as well as domestic cats and dogs. The goal of this study was to survey domestic dogs and cats in Israel for infection with B. persica.MethodsBlood, sera and demographic and clinical data were collected from dogs and cats brought for veterinary care in central Israel. PCR followed by DNA sequencing was used to detect B. persica DNA in blood samples, and an enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies reactive with B. persica antigens in sera from the same animals. This is the first serological survey of B. persica in dogs and the first survey for antibodies reactive with a relapsing fever Borrelia sp. in cats globally.ResultsFour of the 208 dogs (1.9%) and three of 103 cats (2.9%) sampled were positive by PCR for B. persica DNA, and 24 dogs (11.5%) and 18 cats (17.5%) were seropositive for B. persica antigen by ELISA. The ratio between PCR-positivity and seropositivity in both the dog and cat populations was 1:6. All four PCR-positive dogs and two of three PCR-positive cats were seronegative, suggesting a probable recent infection. Thrombocytopenia showed significant association with seropositivity in dogs (P = 0.003). In cats, anemia had a significant association with seropositivity (P = 0.0001), and thrombocytopenia was associated with the combined prevalence of seropositivity or PCR-positivity (P = 0.022).ConclusionsBorrelia persica infection is more prevalent and widespread in domestic canine and feline populations in Israel than previously thought. Dogs and cats may play a role as reservoirs and sentinels for human infection. Precautions should be taken to prevent transfusion-transmitted infection between blood donor and recipient animals.Graphic

Dirofilaria Repens in Dogs and Humans in Slovenia.

IntroductionThe prevalence of Dirofilaria repens in dogs in countries bordering Slovenia ranges from 1.5% to 47.3%. The aim of this study was to estimate its prevalence in Slovenian dogs and to present the cases of dirofilariasis diagnosed in humans from 2010 to 2020.Material and MethodsEpidemiological data were collected and blood samples were taken from 465 dogs older than one year and born in Slovenia. A real-time PCR was performed on all samples to detect filarioid DNA, and a D. repens-and D. immitis-specific real-time PCR was performed on positive samples. Blood samples from 446 dogs were tested for Dirofilaria spp. using a modified Knott’s test. Human cases were diagnosed from histological sections of excised subcutaneous nodules. Descriptive statistics were used to characterise the samples. The one-sample nonparametric chi-squared test was used to assess whether categories of a variable were equally distributed.ResultsThree dogs’ samples tested positive for D. repens using the species-specific real-time PCR, while D. immitis DNA was not detected. The modified Knott’s test was positive in two of the three PCR-positive dogs, two of which had never travelled outside Slovenia’s borders. Four human patients with D. repens dirofilariasis were diagnosed. Since their travel history was unknown, autochthonous transmission could not be confirmed.ConclusionOur study demonstrated a 0.64% prevalence of D. repens infection in dogs in Slovenia. Two cases could be autochthonous.

Genetic diversity of Trypanosoma cruzi parasites infecting dogs in southern Louisiana sheds light on parasite transmission cycles and serological diagnostic performance.

Chagas disease is a neglected zoonosis of growing concern in the southern US, caused by the parasite Trypanosoma cruzi. We genotyped parasites in a large cohort of PCR positive dogs to shed light on parasite transmission cycles and assess potential relationships between parasite diversity and serological test performance. We used a metabarcoding approach based on deep sequencing of T. cruzi mini-exon marker to assess parasite diversity. Phylogenetic analysis of 178 sequences from 40 dogs confirmed the presence of T. cruzi discrete typing unit (DTU) TcI and TcIV, as well as TcII, TcV and TcVI for the first time in US dogs. Infections with multiple DTUs occurred in 38% of the dogs. These data indicate a greater genetic diversity of T. cruzi than previously detected in the US. Comparison of T. cruzi sequence diversity indicated that highly similar T. cruzi strains from these DTUs circulate in hosts and vectors in Louisiana, indicating that they are involved in a shared T. cruzi parasite transmission cycle. However, TcIV and TcV were sampled more frequently in vectors, while TcII and TcVI were sampled more frequently in dogs. These observations point to ecological host-fitting being a dominant mechanism involved in the diversification of T. cruzi-host associations. Dogs with negative, discordant or confirmed positive T. cruzi serology harbored TcI parasites with different mini-exon sequences, which strongly supports the hypothesis that parasite genetic diversity is a key factor affecting serological test performance. Thus, the identification of conserved parasite antigens should be a high priority for the improvement of current serological tests.

Molecular Diagnosis, Prevalence and Importance of Zoonotic Vector-Borne Pathogens in Cuban Shelter Dogs-A Preliminary Study.

The present study aimed to determine the prevalence of zoonotic vector-borne pathogens, including Anaplasma platys, Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Ehrlichia canis and Rickettsia spp. in shelter dogs from Cuba. Blood samples were collected from 100 shelter dogs and examined by molecular methods. Overall, 85 (85%; 95% CI: 77.88–92.12) dogs tested positive for at least one vector-borne pathogen using species-specific qPCR assays. Among the positive samples, E. canis was the most prevalent 62% (95% CI: 52.32–71.68), followed by A. platys 40% (95% CI: 30.23–49.77) and Rickettsia felis 27% (95% CI: 18.15–35.85), whereas 36% (95% CI: 26.43–45.57) showed co-infections. All samples were negative for A. phagocytophilum and B. burgdorferi s.l. The presence of 248 Rhipicephalus sanguineus ticks collected from the dogs was not statistically associated with the occurrence of infections. Thrombocytopenia was the most frequent haematological alteration found in PCR-positive dogs; it was statistically associated with the presence of E. canis, as well as co-infections (p < 0.05). The phylogenetic analyses of A. platys and E. canis based on 16S rRNA, groEL and gltA genes showed a low genetic diversity between Cuban strains. The present study demonstrates the high prevalence of vector-borne pathogens with zoonotic potential in shelter dogs from Cuba.

Molecular detection and characterization of Hepatozoon canis in stray dogs from Cuba.

Canine hepatozoonosis caused by Hepatozoon canis is a worldwide distributed tick-borne disease of domestic and wild canids that is transmitted by ingestion of Rhipicephalus sanguineus sensu lato (s.l.) ticks. The present study was aimed to determine the prevalence of Hepatozoon infections in 80 stray dogs from Havana Province in Cuba, and to confirm the species identity and phylogenetic relationships of the causative agent. Samples were screened by microscopical examination of thin blood smears for the presence of Hepatozoon spp. gamonts and by genus-specific SYBR green-based real-time PCR assay targeting the 18S rRNA gene. Direct microscopy examination revealed Hepatozoon gamonts in the peripheral blood of 8 dogs (10.0%; 95% CI: 4.80-18.0%), while 38 animals (47.5%; 95% CI: 36.8-58.4%) were PCR-positive, including all microscopically positive dogs. Hence, the agreement between the two detection methods was 'poor' (κ = 0.20). Hematological parameters did not differ significantly between PCR-positive and PCR-negative dogs (p > 0.05). The DNA sequences of the 18S rRNA gene of the Hepatozoon spp. from Cuban dogs showed a nucleotide identity >99% with those of 18S rRNA sequences of Hepatozoon canis isolates from Czech Republic, Brazil and Spain. Phylogenetic analysis revealed that obtained sequences clustered within the Hepatozoon canis clade, different from the Hepatozoon felis or Hepatozoon americanum clades. The present study represents the first molecular characterization of Hepatozoon canis in stray dogs within Cuba.

Evidence of acute phase reaction in asymptomatic dogs naturally infected with Babesia canis

Asymptomatic outdoor dogs can be carriers of Babesia canis, but data describing the development of an acute phase response (APR) are not available. We hypothesised that these dogs have a moderate APR that could be detected by hematological and biochemical changes. Two groups of Babesia-exposed dogs were represented by nine B. canis PCR-positive and twenty B. canis PCR-negative, seroreactive dogs. The control group consisted of ten Babesia-naïve dogs. Serum amyloid A (SAA), paraoxonase-1 (PON-1), complete blood count, and biochemistry parameters were analysed by standard methodologies. Protein and lipoprotein fractions were separated using agarose gel electrophoresis (GE), and the dominant diameters of lipoproteins were assessed on gradient GE. Results were evaluated using non-parametric tests and the Receiver Operating Characteristic curve. SAA (median 39.0 μg/mL, range 2.2–48.8 μg/mL), total protein (median 74.7 g/L, range 57.1–98.3 g/L) and the dominant diameter of α-lipoproteins (median 13.31 nm, range 12.09–14.17 nm) in B. canis PCR-positive dogs were higher relative to dogs in the control group or dogs that were PCR-negative but seroreactive (p < 0.001 for both groups). Mild to moderate anemia (4/29), thrombocytopenia (7/29), and leukocyte counts that were close to the upper limit of the reference range were encountered in both Babesia-exposed groups. When compared to controls, Babesia-exposed dogs displayed decreased a PON-1 activity and protein GE pattern consistent with low-grade chronic inflammation (p < 0.001 for both groups). Dogs with detectable amounts of B. canis DNA in blood contain increased levels of SAA and total protein along with α-lipoproteins that display an increased diameter relative to those dogs with positive Babesia serology but undetectable levels of B. canis DNA in blood.

Leishmania infection in cats and dogs housed together in an animal shelter reveals a higher parasite load in infected dogs despite a greater seroprevalence among cats

BackgroundAn outbreak of leishmaniosis was studied in cats and dogs housed together with no separation in an animal shelter in Israel.MethodsThe study included recording of clinical signs, serology for Leishmania infection by ELISA, PCR of blood for Leishmania DNA by ITS1 HRM and kDNA PCR, parasite quantification, and trapping of sand flies around the shelter.ResultsThirty-seven % (22/60) of the dogs and 75% (50/67) of the cats were seropositive to L. infantum with a significantly higher seropositivity rate in the cat population (χ2 = 42.160, P < 0.0001). Twenty-five percent (15/60) of the dogs were positive for Leishmania by blood PCR, 12% by the Leishmania ITS1 HRM PCR and 22% by kDNA PCR. Of the cats, 16% (11/67) were positive by kDNA PCR and none by ITS1 HRM PCR. All the PCR-positive animals were infected by L. infantum verified by DNA sequencing and there was no significant difference between the PCR-positivity in the dog and cat populations. Altogether, 43% (26/60) of the dogs and 79% (53/67) of the cats were positive by serology or PCR for L. infantum. The average Leishmania parasite load in the blood of PCR-positive dogs (42,967 parasites/ml) was significantly higher than in PCR-positive cats (1259 parasites/ml) (t(12) = 2.33, P = 0.037). Dogs that were positive by the Leishmania ITS1 HRM PCR and kDNA PCR had significantly higher parasite loads than dogs positive only by the kDNA PCR (t(11) = − 3.186580, P < 0.009). No significant effect was found for FIV seropositivity on Leishmania infection in the cats (χ2 = 0.506, P = 0.777). A higher percentage of Leishmania-positive dogs showed clinical signs compatible with leishmaniosis compared to Leishmania-positive cats (100 vs 52.8%, χ2 =15.242, P < 0.0001). Phlebotomus perfiliewi, a proven vector of L. infantum, comprised 92% of trapped sand flies.ConclusionsComparisons of populations of cats and dogs exposed to sand flies and L. infantum under the same conditions indicated that although a high rate of exposure was detected in cats as manifested by a significantly greater degree of seropositivity, dogs had significantly higher blood parasite loads, and were likely to be more infectious to sand flies than cats.

Ehrlichia canis TRP36 diversity in naturally infected-dogs from an urban area of Colombia

Ehrlichia canis is the etiologic agent of a highly prevalent tick-borne disease, canine monocytic ehrlichiosis (CME). Four defined E. canis genotypes based on the trp36 gene sequences have been reported, three of them identified in North or South America. The diversity of E. canis has been investigated using genetic and serologic approaches based on distinct 36 kDa tandem repeat protein (trp36) gene sequences that have been reported. The main objectives of this study were to determine the prevalence of E. canis infection in dogs from Medellín, Colombia by PCR and determine the E. canis diversity using molecular and serologic approaches. Blood was collected from dogs (n = 300) with clinical signs of CME for PCR detection of E. canis 16S rRNA, dsb and trp36 DNA. Phylogenetic analysis of trp36 gene sequences was performed using MEGA. A serological evaluation was performed using immunofluorescence microscopy and ELISA with species-specific peptides from E. canis TRP19 and TRP36 (3 genotypes) and E. chaffeensis (TRP32). E. canis DNA (16S rRNA and/or dsb) was detected in 18 % (53/300) of dogs by PCR amplification. The trp36 gene was amplified and sequenced from 35/53 16S rRNA/dsb PCR positive samples revealing three genotypes: United States (US; n = 21), Costa Rica (CR; n = 11), and Brazil (BR; n = 3). Most dogs (33/35) with detectable trp36 DNA had anti-E. canis TRP19 and TRP36 peptide antibodies that corresponded to the genotype detected by PCR. Dogs that had antibodies to the TRP19 peptide (82/300; 38 %), also had antibodies to one or more genotype-specific TRP36 peptides. Based on TRP36 serology, the dogs exhibited highest frequency of infection with the US genogroup (US = 26), followed by the CR genogroup (CR = 19) and the BR genogroup (BR = 11). Notably, 26/53 trp36 PCR positive dogs had detectable antibodies to multiple E. canis genotypes (US/BR/CR = 8, BR/CR = 7, US/CR = 6 and US/BR = 5) suggesting coinfection or multiple sequential infections with different genotypes. Colombian dogs did not have antibodies to E. chaffeensis as determined by a TRP32 species-specific ELISA. Our results demonstrate the presence of three previously defined genotypes in North and South America in Colombian dogs (US, BR, CR). These results also demonstrate that TRP19 and TRP36 serology can provide valuable information regarding E. canis exposure and the potential genotype(s) involved in infection.

High prevalence of Trypanosoma cruzi infection in shelter dogs from southern Louisiana, USA

BackgroundChagas disease is a zoonotic disease caused by the protozoan parasite Trypanosoma cruzi. The role of dogs as sentinels has been proposed in multiple regions, as they are a domestic reservoir for T. cruzi. Our objective was to determine the prevalence of T. cruzi infection in shelter dogs from southern Louisiana, and assess its magnitude and distribution.ResultsA total of 540 dogs were enrolled, from 20 animal shelters, and tested for T. cruzi infection by serological tests (rapid test, ELISA and western blot) and PCR. We documented a high prevalence of T. cruzi infection with at least 6.9% (95% CI: 5.0–9.3%) seropositive and 15.7% (95% CI: 12.9–19.1%) PCR-positive dogs. Serological tests showed limited agreement, and concordance between serology and PCR was higher when considering reactivity to single serological tests. Trypanosoma cruzi infection was distributed evenly among shelters. Infection was significantly correlated with age (R2 = 0.99), indicating an incidence of new cases of 2.27 ± 0.25% per year.ConclusionTrypanosoma cruzi infection is a significant and widespread veterinary problem in shelter dogs in the region, although it is mostly unnoticed by health professionals. This highlights the need for greater awareness of T. cruzi infection among the veterinary community and dog owners.

Diagnosis of acute canine leptospirosis using multiple laboratory tests and characterization of the isolated strains

BackgroundDogs presenting with acute leptospirosis may present non-specific clinical and laboratory findings, and the definitive diagnosis may require additional confirmatory tests, including bacterial culture, for the direct or indirect identification of the pathogen. The present study describes the diagnosis of leptospirosis in suspected dogs based on the use of multiple diagnostic tests, including serological, molecular and bacteriological tests, along with the characterization of the recovered leptospiral strains.ResultsUrine, serum and blood samples were collected from 33 dogs with suspected clinical leptospirosis treated at the University of São Paulo Veterinary Hospital Service (Hovet FMVZ-USP) between 2013 and 2016. Only dogs with high blood urea nitrogen and creatinine levels in association with multiple clinical manifestations of the disease were included. Leptospiral culture, PCR and serology (Microscopic agglutination test - MAT) were performed in blood and urine samples taken from all suspected dogs at clinical presentation, and an additional prospective MAT titration was performed in seven dogs. Infection could be identified exclusively by PCR in 10 dogs (30.3%), exclusively by MAT in four dogs (12.1%) and by both tests in four dogs, totaling 18 dogs (54.5–95%CI: 37.6–71.5). Six out of eight MAT-confirmed cases presented with the highest titers against the Icterohaemorrhagiae serogroup. Leptospires were recovered from urine samples from two PCR-positive dogs, and both strains could be characterized by Multilocus Sequence Analysis and serogrouping as L. interrogans serogroup Icterohaemorrhagiae. Both isolates were shown to be pathogenic in the hamster model.ConclusionsThe simultaneous use of MAT and PCR was able to increase the diagnosis of leptospirosis in clinically suspected cases. Despite the increasing incidence of new serovars affecting dogs being reported in different locations, our results suggest that leptospiral strains belonging to the Icterohaemorrhagiae serogroup are still a major causative agent of canine leptospirosis in São Paulo, Brazil.