Serosurvey study of Toscana virus in domestic animals, Granada, Spain

Toscana virus (TOSV) is transmitted by infected sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis in humans. It remains unclear if there are animal reservoirs able to maintain the virus through the cold months of the year, when the vector is not circulating. From May to October of 2006 and 2007, we conducted a serosurvey study on domestic animals from Granada province (southern Spain). TOSV was investigated in 1186 serum samples from horses, goats, pigs, cats, dogs, sheep, and cows by serology (indirect fluorescence assay), viral culture, and RT-polymerase chain reaction. Specific anti-TOSV antibodies were detected in 429 (36.2%) serum samples. The highest seropositivity rates were observed in cats (59.6%) and dogs (48.3%). These results suggest that an important percentage of the domestic animals have been infected by TOSV. Significantly different seroprevalence rates were detected in goats among distinct geographical areas. All viral cultures were negative. TOSV was detected by RT-polymerase chain reaction in only one serum sample from a goat. Thus, the studied animals do not seem to act as reservoirs for TOSV; otherwise, they could be amplifying hosts for the virus.     

Evaluation of three recombinant Leishmania infantum antigens in human and canine visceral leishmaniasis diagnosis

Visceral leishmaniasis (VL) is a neglected disease and is fatal if untreated. Dogs serve as reservoirs for Leishmania infantum (syn. L. chagasi) due to their susceptibility to infection and high skin parasitism. Therefore, VL control in Brazil involves the elimination of seropositive dogs, among other actions. However, the most frequently used serological tests have limitations regarding sensitivity and specificity. In this study, we have selected three Leishmania antigens (C1, C8 and C9) and have produced them as recombinant proteins using pET-28a-TEV vector and Escherichia coli BL-21 as expression system. When tested in ELISA with human samples, the C9 antigen was the one showing the most promising results, with 68% sensitivity and 78% specificity. When testing canine samples, the C1, C8 and C9 antigens showed a sensitivity range from 70% to 80% and specificity range from 60% to 90%. The C1 antigen presented higher sensitivity (80%) and the C8 antigen presented higher specificity (90%). Due to it, we decided to mix and test C1 and C8 antigens together, resulting in the C18 antigen. The mix also yielded high percentages of detected symptomatic and asymptomatic dogs however it did not improve the performance of the diagnostic. Comparison of our tests with the tests recommended by the Brazilian Ministry of Health revealed that our antigens’ sensitivities and the percentage of detected asymptomatic dogs were much higher. Our results suggest that the C1, C8, C18 and C9 recombinant proteins are good antigens to diagnose canine visceral leishmaniasis and could potentially be used in screening tests. To diagnose human visceral leishmaniasis, the C9 antigen presented reasonable results, but more optimization must be performed for this antigen to provide better performance.     

Serological evidences of canine brucellosis as a new emerging disease in Iran

ObjectiveTo detect the evidences of significant exposure of dogs with Brucella canis (B. canis) and other Brucella species in southeast of Iran by serological assays.MethodsBlood samples were prepared from 62 privately owned and 33 kenneled dogs and screened by indirect immunoflorescence assay for B. canis. Rose Bengal test (RBT) and standard tube test (SAT) or wright test were used for screening the other Brucella species.ResultsSerological evidences of exposure to B. canis were found in 15.8% of the studied dogs, whereas RBT and SAT showed 29.5% and 12.6% seropositivity, respectively.ConclusionsThis study shows that B. canis is endemic in southeastern of Iran and canine brucellosis can be considered as a new emerging disease. Moreover, our results demonstrate that dogs will be at high risk in brucellosis-infected farms. Therefore, further investigations must be performed to evaluate the seroprevalence and zoonotic potential of this disease in different geographical parts of Iran.     

Hepadnavirus DNA Is Detected in Canine Blood Samples in Hong Kong but Not in Liver Biopsies of Chronic Hepatitis or Hepatocellular Carcinoma

Chronic hepatitis and hepatocellular carcinoma (HCC) caused by the hepadnavirus hepatitis B virus (HBV) are significant causes of human mortality. A hepatitis-B-like virus infecting cats, domestic cat hepadnavirus (DCH), was reported in 2018. DCH DNA is hepatotropic and detectable in feline blood or serum (3.2 to 12.3%). Detection of HBV DNA has been reported in sera from 10% of free-roaming dogs in Brazil, whereas 6.3% of sera from dogs in Italy tested positive for DCH DNA by real-time quantitative PCR (qPCR). If DCH, HBV, or another hepadnavirus is hepatotropic in dogs, a role for such a virus in the etiology of canine idiopathic chronic hepatitis (CH) or HCC warrants investigation. This study investigated whether DCH DNA could be detected via qPCR in blood from dogs in Hong Kong and also whether liver biopsies from dogs with confirmed idiopathic CH or HCC contained hepadnaviral DNA using two panhepadnavirus conventional PCRs (cPCR) and a DCH-specific cPCR. DCH DNA was amplified from 2 of 501 (0.4%) canine whole-blood DNA samples. A second sample taken 6 or 7 months later from each dog tested negative in DCH qPCR. DNA extracted from 101 liver biopsies from dogs in Hong Kong or the USA, diagnosed by board-certified pathologists as idiopathic CH (n = 47) or HCC (n = 54), tested negative for DCH DNA and also tested negative using panhepadnavirus cPCRs. This study confirms that DCH DNA can be detected in canine blood by qPCR, although at a much lower prevalence than that reported previously. We identified no evidence to support a pathogenic role for a hepadnavirus in canine idiopathic CH or HCC.     

Molecular diagnosis of canine visceral leishmaniasis: Identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR

The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dye-based system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmaniainfantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples.     

The first reported cases of canine schistosomiasis mekongi in Cambodia

We have been conducting surveys of schistosomiasis mekongi along the Mekong river in Cambodia since 1997. We attempted to detect canine schistosome infection during the survey in 2000 because dogs were reported to be natural reservoirs of the Mekong schistosome in Lao PDR. A total of 28 canine fecal samples were collected in Kbal Chuor village, Kratie Province and examined for schistosome eggs. One specimen had schistosome eggs (positive rate = 3.6%; egg density = 100/gram stool), which showed characteristics of Schistosoma mekongi. During the 2001 survey, one out of 310 canine stool samples was positive for schistosome eggs (positive rate = 0.32%; egg density = 3,456/gram stool). These are the first confirmed cases of canine schistosomiasis mekongi in Cambodia, which suggests that dogs are animal reservoirs of S. mekongi in the survey area. We further tried to detect S. mekongi in cows, water buffalos, pigs,horses, and field rats in five villages in Kratie Province; no schistosome egg was found in the stools of these animals.     

Domestic dog health worsens with socio-economic deprivation of their home communities

Dogs play an important role in infectious disease transmission as reservoir hosts of many zoonotic and wildlife pathogens. Nevertheless, unlike wildlife species involved in the life cycle of pathogens, whose health status might be a direct reflection of their fitness and competitive abilities, dog health condition could be sensitive to socio-economic factors impacting the well-being of their owners. Here, we compare several dog health indicators in three rural communities of Panama with different degrees of socio-economic deprivation. From a total of 78 individuals, we collected blood and fecal samples, and assessed their body condition. With the blood samples, we performed routine hematologic evaluation (complete blood counts) and measured cytokine levels (Interferon-γ and Interleukin-10) through enzyme-linked immunosorbent assays. With the fecal samples we diagnosed helminthiases. Dogs were also serologically tested for exposure to Trypanosoma cruzi and canine distemper virus, and molecular tests were done to assess T. cruzi infection status. We found significant differences between dog health measurements, pathogen prevalence, parasite richness, and economic status of the human communities where the dogs lived. We found dogs that were less healthy, more likely to be infected with zoonotic pathogens, and more likely to be seropositive to canine distemper virus in the communities with lower economic status. This study concludes that isolated communities of lower economic status in Panama may have less healthy dogs that could become major reservoirs in the transmission of diseases to humans and sympatric wildlife.     

Identification of Bartonella Infections in Febrile Human Patients from Thailand and Their Potential Animal Reservoirs

To determine the role of Bartonella species as causes of acute febrile illness in humans from Thailand, we used a novel strategy of co-cultivation of blood with eukaryotic cells and subsequent phylogenetic analysis of Bartonella-specific DNA products. Bartonella species were identified in 14 blood clots from febrile patients. Sequence analysis showed that more than one-half of the genotypes identified in human patients were similar or identical to homologous sequences identified in rodents from Asia and were closely related to B. elizabethae, B. rattimassiliensis, and B. tribocorum. The remaining genotypes belonged to B. henselae, B. vinsonii, and B. tamiae. Among the positive febrile patients, animal exposure was common: 36% reported owning either dogs or cats and 71% reported rat exposure during the 2 weeks before illness onset. The findings suggest that rodents are likely reservoirs for a substantial portion of cases of human Bartonella infections in Thailand.     

PARTICIPATION OF TICKS IN THE INFECTIOUS CYCLE OF CANINE VISCERAL LEISHMANIASIS,IN TERESINA,PIAUí, BRAZIL

In this study, we detected Leishmania spp. infection in R. sanguineus collected from dogs that were naturally infected with L. (L.) infantum. We examined 35 dogs of both sexes and unknown ages. The infected dogs were serologically positive by the immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and Quick Test-DPP (Dual Path Platform), as well as parasitological examination of a positive skin biopsy or sternal bone marrow aspiration. Ten negative dogs were included as controls. The ticks that infested these dogs were collected in pools of 10 adult females per animal. The PCR was performed with specific primers for Leishmania spp., which amplified a 720-bp fragment. Of the 35 analyzed samples, a product was observed in eight samples (8/35; 22.9%). We conclude that the presence of parasite DNA suggests that ticks participate in the zoonotic cycle of canine visceral leishmaniasis, in the city of Teresina, Piauí.     

Recombinant Leishmania (Leishmania) infantum Ecto-Nucleoside Triphosphate Diphosphohydrolase NTPDase-2 as a new antigen in canine visceral leishmaniasis diagnosis

Canine visceral leishmaniasis is an important public health concern. In the epidemiological context of human visceral leishmaniasis, dogs are considered the main reservoir of Leishmania parasites; therefore, dogs must be epidemiologically monitored constantly in endemic areas. Furthermore, dog to human transmission has been correlated with emerging urbanization and increasing rates of leishmaniasis infection worldwide. Leishmania (Leishmania) infantum (L. chagasi) is the etiologic agent of visceral leishmaniasis in the New World. In this work, a new L. (L.) infantum (L. chagasi) recombinant antigen, named ATP diphosphohydrolase (rLic-NTPDase-2), intended for use in the immunodiagnosis of CVL was produced and validated. The extracellular domain of ATP diphosphohydrolase was cloned and expressed in the pET21b-Escherichia coli expression system. Indirect ELISA assays were used to detect the purified rLic-NTPDase-2 antigen using a standard canine sera library. This library contained CVL-positive samples, leishmaniasis-negative samples and samples from Trypanosoma cruzi-infected dogs. The results show a high sensitivity of 100% (95% CI = 92.60–100.0%) and a high specificity of 100% (95% CI = 86.77–100.0%), with a high degree of confidence (k = 1). These findings demonstrate the potential use of this recombinant protein in immune diagnosis of canine leishmaniasis and open the possibility of its application to other diagnostic approaches, such as immunochromatography fast lateral flow assays and human leishmaniasis diagnosis.     

Isolation and identification of thermophilic Campylobacter species in faecal samples from Swedish dogs

To investigate the role of Swedish dogs as potential reservoirs of thermophilic Campylobacter species, faecal samples were analysed from 91 dogs in 2001. The majority of dogs (n = 84) were healthy family dogs. Campylobacter spp. were isolated from 51 of the 91 dogs (56%). A significant difference in isolation rates was observed between younger and older dogs: 76% of the younger dogs (5-12 months) were positive, compared with 39% of dogs > or = 13 months (p < 0.01). Two different selective media, Preston and CAT, were used for isolation of Campylobacter species. 104 Campylobacter isolates were identified to species level using polymerase chain reaction and restriction enzyme analysis techniques. Campylobacter upsaliensis predominated and was isolated from 39 dogs, C. jejuni from 10, C. coli from 2, C. helveticus from 2 and C. lari from 1 dog. Four dogs had mixed flora with 2 different Campylobacter species. These data clearly show that younger dogs in particular frequently shed thermophilic Campylobacter spp, which could be of impact for public health. To establish the zoonotic potential of canine Campylobacter isolates, both human and canine isolates have to be further characterized and compared.     

Comparison of PCR with stained slides of bone marrow and lymph nodes aspirates with suspect diagnosis for leishmaniasis

Visceral leishmaniasis (VL), also known as kala-azar, is a disseminated protozoan infection caused by Leishmania donovani complex. Traditionally the definite diagnosis is made by amastigote detection in the tissue. The aim this study was to evaluate the PCR technique in stained slides of bone marrow and lymph nodes aspirates with suspect diagnosis for leishmaniasis. Slides were selected totaling 62 suspect cases (33 bone marrow samples and 29 lymph node samples) and 17 positive cases (8 bone marrow and 9 lymph node). From 62 suspect cases, 39 (62.90%) were confirmed to be positive being 17 (n = 29) lymph node aspirates and 22 (n = 33) bone marrow. This finding is in agreement with the higher sensitivity of the PCR assay compared to direct microscopic observation. In conclusion, the findings of this study supports the use of PCR on archive cytological preparation stained slides for the diagnosis of canine visceral leishmaniasis, emphasizing the higher sensitivity of this technique when compared to direct microscopic examination and mostly the use of the suspect status for the cytology samples that presents the previously mentioned particularities with focus on detecting the oligosymptomatic or assymptomatic dogs in endemic areas functioning as potential reservoirs for this disease.     

Molecular identification of Neospora caninum from calf/foetal brain tissue and among oocysts recovered from faeces of naturally infected dogs in southern Ethiopia

This study sought to confirm and investigate further recently published information regarding the occurrence of Neospora caninum in cattle in Ethiopia and investigate infection in dogs, the canine definitive host, in this region. Faecal samples from 383 dogs in Hawassa, Ethiopia were examined by microscopy for Neospora-like oocysts, and positive samples then analysed by a molecular approach (DNA isolation, PCR and sequencing at the ITS1 gene). Brain tissue samples from four late term aborted foetuses, one congenitally defective calf (hind leg arthrogryposis) and placental tissue from cattle in the same area were also examined by the same molecular approach. All foetal, calf and placental tissue were associated with Neospora seropositive dams. A high prevalence of Neospora-like oocysts (11.5 μm ± 1.5 μm diameter) was observed in faecal samples from dogs (37 positive samples; 9.7% prevalence), and in 17 of these the identification was confirmed by PCR, giving a prevalence of confirmed infection of 4.4%. N. caninum DNA was also detected in all foetal and calf brain tissue samples. Sequencing revealed only minor differences among all PCR products, whether from oocysts or from brain tissue samples. These data provide molecular evidence of the presence of N. caninum infection in both dog and cattle in this region of Ethiopia. Moreover these findings highlight the role of dogs in maintaining and spreading the infection horizontally in the study area. The high frequency of N. caninum infection in household dogs as well as farm dogs is worthy of further investigation.     

Anti-platelet antibodies in a natural animal model of sulphonamide-associated thrombocytopaenia

Delayed hypersensitivity (HS) reactions to sulphonamide antimicrobials occur in both humans and dogs with a similar clinical presentation, and may include thrombocytopaenia. Drug-dependent anti-platelet antibodies have been identified in humans with sulphonamide-associated thrombocytopaenia. Our purpose was to determine whether similar antibodies were present in dogs with sulphonamide-associated thrombocytopaenia.

Flow cytometry was used to detect anti-platelet antibodies in sera from 32 dogs with sulphonamide HS, eight dogs that tolerated sulphonamide therapy without adverse reactions and nine healthy control dogs were used as controls. Anti-platelet antibodies were found more frequently, with significantly stronger fluorescence signals, in HS dogs (75%) compared to ‘tolerant’ dogs (38%), and in HS dogs with thrombocytopaenia (90%) compared to HS dogs with normal platelet counts (46%). Binding to platelets was enhanced in the presence of soluble sulphonamide in 42% of positive samples. Experiments with canine Glanzmann's platelets, and competition assays with fibrinogen fragments or anti-GP antibodies, did not support the hypothesis that these canine antibodies target the fibrinogen receptor. In conclusion, anti-platelet antibodies were identified in dogs with sulphonamide-associated thrombocytopaenia, which suggests a similar immunopathogenesis for this reaction in dogs as seen in humans. Further work in both species will determine whether these antibodies are pathogenic in vitro.     

Independent evaluation of a canine Echinococcosis Control Programme in Hobukesar County,Xinjiang, China

The Xinjiang Uyghur Autonomous Region in northwest China is one of the world's most important foci for cystic echinococcosis. Domestic dogs are the main source for human infection, and previous studies in Xinjiang have found a canine Echinococcus spp. coproELISA prevalence of between 36% and 41%. In 2010 the Chinese National Echinococcosis Control Programme was implemented in Xinjiang, and includes regular dosing of domestic dogs with praziquantel. Six communities in Hobukesar County, northwest Xinjiang were assessed in relation to the impact of this control programme through dog necropsies, dog Echinococcus spp. coproantigen surveys based on Lot Quality Assurance Sampling (LQAS) and dog owner questionnaires. We found that 42.1% of necropsied dogs were infected with Echinococcus granulosus, and coproELISA prevalences were between 15% and 70% in the communities. Although approximately half of all dog owners reported dosing their dogs within the 12 months prior to sampling, coproELISA prevalence remained high. Regular praziquantel dosing of owned dogs in remote and semi-nomadic communities such as those in Hobukesar County is logistically very difficult and additional measures should be considered to reduce canine echinococcosis.     

Prevalence of intestinal helminthes in owned dogs in Kerman city, Iran

ObjectiveTo survey the prevalence of canine gastrointestinal helminthes in dogs presented to the Veterinary faculty of the University of Kerman between May and November 2011.MethodsA total of 70 fecal samples were evaluated by the fecal sedimentation method.ResultsThe prevalence of gastrointestinal helminthes was 7.14%. The parasites most frequently detected were Toxocara canis (T. canis) (4.3%); Toxascaris leonina (T. leonina) (1.4%) and Teania spp. (1.4%). The age distribution of intestinal parasites in dogs showed that the dog less than 1 year old had a higher overall prevalence than those dogs over 12 months of age but there was not significant (P>0.05). Also there was no significant difference in the prevalence between male (7.7%) and female (6.5%) dogs (P>0.05).ConclusionsIt is thought that the reduction in the frequency of the dogs with those helminthes may be mainly a result of the improvement in breeding environment and the routine use of antihelmintics. The significance of zoonotic diseases caused by intestinal helminthes makes it necessary for us to know the infection status of domestic dogs and to take measures for further control. It is concluded that veterinarians have an important role in educating dog owners of these potential risks and means for preventing or minimizing zoonotic transmission.     

Thirteen Years of Phleboviruses Circulation in Lombardy,a Northern Italy Region

Phleboviruses transmitted by phlebotomine sandflies are endemic in the Mediterranean basin. Toscana phlebovirus (TOSV), Sicilian phlebovirus (SFSV), and Naples phlebovirus (SFNV) are responsible of summer fever, with well-known pathogenic potential for humans ranging from asymptomatic to mild fever, in addition to neuro-invasive infections during summer. Although TOSV, in particular, is a significant and well-known human pathogen, SFVs remain neglected, with many gaps in the relevant knowledge. Sero-epidemiological studies and case reports recently showed a geographical wider distribution than previously considered, although the real incidence of phleboviruses infections in the Mediterranean area is still unknown. Here we retrospectively evaluated the circulation of phleboviruses during summer seasons between 2007 and 2019 in 649 patients showing neurological symptoms using both molecular and serological approaches. We found that 42/649 (6.5%) subjects experienced phlebovirus infection and only 10/42 cases were detected by molecular assays, whereas the other 32/42 were identified using serological approaches, including neutralization assays. During the 2013 summer, an outbreak in the Lombardy region is described because the prevalence of phlebovirus infection reached 37.2% (19/51 subjects). Interestingly, only 5/19 (26.5%) reported traveling in endemic areas. Of note, no cross-neutralization was observed between different strains tested, showing the possibility to be reinfected by newly discovered phlebovirus strains. In conclusion, phlebovirus infections are still inadequately considered by physicians and are generally underestimated. However, based on our results, sandfly fever viruses should be routinely included in diagnostic panels during summer period, including in Northern Italy.     

2012-2018年内蒙古自治区犬棘球绦虫感染空间分布特征

目的 了解2012-2018年内蒙古自治区犬棘球绦虫感染分布规律及变化趋势,为重点地区棘球蚴病防控提供参考依据.方法 收集2012-2018年内蒙古自治区棘球蚴病流行区犬粪棘球绦虫感染数据和人群棘球蚴病抽样调查数据,对犬棘球绦虫感染率及人群棘球蚴病患病率进行分析;采用空间自相关分析方法对犬棘球绦虫感染空间分布特征及聚集...     

The transmission dynamics of canine American cutaneous leishmaniasis in Huánuco, Peru

The epidemiology of canine American cutaneous leishmaniasis (ACL) due to Leishmania (Viannia) spp. was investigated in Huánuco, Peru to 1) describe the natural course of canine L. (Viannia) infections and 2) assess the role of domestic dogs as ACL reservoir hosts. Over a three-year period 1,022 dogs were surveyed, with cumulative village L. (Viannia) prevalence being 26% (range = 0-100%). The incidence of L. (Viannia) was estimated to be 0.285 dogs/year (95% confidence interval [CI] = 0.160-0.410) using cross-sectional data and 0.291 dogs/year (95% CI = 0.195-0.387) using data from 108 dogs that were surveyed prospectively. The recovery rate was estimated to be 0.456 dogs/year (95% CI = 0.050-0.862) and 0.520 dogs/year (95% CI = 0.302-0.738), respectively. Using those findings, the basic reproduction number was estimated to be R0 approximately to 1.9; if dogs were the principal ACL reservoirs, the mean yearly effort (i.e., coverage or elimination) of a dog control intervention (e.g., collaring, culling, or vaccination) to ensure the elimination of L. (Viannia) spp. transmission would be as low as 47%.     

Update in Diagnostics of Toscana Virus Infection in a Hyperendemic Region (Southern Spain)

The sandfly fever Toscana virus (TOSV, genus Phlebovirus, family Phenuiviridae) is endemic in Mediterranean countries. In Spain, phylogenetic studies of TOSV strains demonstrated that a genotype, different from the Italian, was circulating. This update reports 107 cases of TOSV neurological infection detected in Andalusia from 1988 to 2020, by viral culture, serology and/or RT-PCR. Most cases were located in Granada province, a hyperendemic region. TOSV neurological infection may be underdiagnosed since few laboratories include this virus in their portfolio. This work presents a reliable automated method, validated for the detection of the main viruses involved in acute meningitis and encephalitis, including the arboviruses TOSV and West Nile virus. This assay solves the need for multiple molecular platforms for different viruses and thus, improves the time to results for these syndromes, which require a rapid and efficient diagnostic approach.