Follow-up monitoring in a cat with leishmaniosis and coinfections with Hepatozoon felis and ‘Candidatus Mycoplasma haemominutum’

Case summary A 6-year-old female neutered domestic shorthair cat from Cyprus was presented with multiple ulcerated skin nodules. Cytology and histopathology of the lesions revealed granulomatous dermatitis with intracytoplasmic organisms, consistent with amastigotes of Leishmania species. Biochemistry identified a mild hyperproteinaemia. Blood extraction and PCR detected Leishmania species, Hepatozoon species and ‘Candidatus Mycoplasma haemominutum’ (CMhm) DNA. Subsequent sequencing identified Hepatozoon felis. Additionally, the rRNA internal transcribed spacer 1 locus of Leishmania infantum was partially sequenced and phylogeny showed it to cluster with species derived from dogs in Italy and Uzbekistan, and a human in France. Allopurinol treatment was administered for 6 months. Clinical signs resolved in the second month of treatment with no deterioration 8 months post-treatment cessation. Quantitative PCR and ELISA were used to monitor L infantum blood DNA and antibody levels. The cat had high L infantum DNA levels pretreatment that gradually declined during treatment but increased 8 months post-treatment cessation. Similarly, ELISA revealed high levels of antibodies pretreatment, which gradually declined during treatment and increased slightly 8 months post-treatment cessation. The cat remained PCR positive for CMhm and Hepatozoon species throughout the study. There was no clinical evidence of relapse 24 months post-treatment. Relevance and novel information To our knowledge, this is the first clinical report of a cat with leishmaniosis with H felis and CMhm coinfections. The high L infantum DNA levels post-treatment cessation might indicate that although the lesions had resolved, prolonged or an alternative treatment could have been considere

Prioritization of Companion Animal Transmissible Diseases for Policy Intervention in Europe

A number of papers have been published on the prioritization of transmissible diseases in farm animals and wildlife, based either on semiquantitative or truly quantitative methods, but there is no published literature on the prioritization of transmissible diseases in companion animals. In this study, available epidemiological data for diseases transmissible from companion animals to man were analysed with the aim of developing a procedure suitable for their prioritization within a European framework. A new method and its associated questionnaire and scoring system were designed based on methods described by the World Organisation for Animal Health (OIE). Modifications were applied to allow for the paucity of specific information on companion animal transmissible diseases. The OIE method was also adapted to the subject and to the regional scope of the interprofessional network addressing zoonotic diseases transmitted via companion animals in Europe: the Companion Animals multisectoriaL interprofessionaL Interdisciplinary Strategic Think tank On zoonoses (CALLISTO). Adaptations were made based on information collected from expert groups on viral, bacterial and parasitic diseases using a structured questionnaire, in which all questions were closed-ended. The expert groups were asked to select the most appropriate answer for each question taking into account the relevance and reliability of the data available in the scientific literature. Subsequently, the scoring of the answers obtained for each disease covered by the questionnaire was analysed to obtain two final overall scores, one for human health impact and one for agricultural economic impact. The adapted method was then applied to select the 15 most important pathogens (five for each pathogen group: viral, bacterial and parasitic) on the basis of their overall impact on public health and agriculture. The result of the prioritization exercise was a joint priority list (available at www.callistoproject.eu) of relevant pathogens according to these two criteria. As the scope of CALLISTO was comprehensive in terms of geographical area, animal species involved and impact of the diseases, the list of prioritized diseases had to accommodate the realities in different European countries and the differences in biology and animalehuman relationships in a wide range of species including cats and dogs, pet pigs and sheep as well as captive reptiles. The methodology presented in this paper can be used to generate accurate priority lists according to narrower and more specific objectives

Leishmania infantum-specific IFN-γ production in stimulated blood from dogs with clinical leishmaniosis at diagnosis and during treatment

There is limited data regarding Leishmania infantum specific T cell mediated immunity in naturally infected sick dogs at the time of diagnosis and during anti-Leishmania treatment. Our aim was to investigate the kinetics of L. infantum specific IFN-γ production in dogs with leishmaniosis at the time of diagnosis and during treatment and to correlate it with specific L. infantum antibodies, blood parasitemia and clinicopathological findings. Thirty-four dogs were diagnosed with leishmaniosis based on physical examination, routine laboratory tests and L. infantum-specific antibody levels by quantitative ELISA. Heparinized whole blood was stimulated with L. infantum soluble antigen (LSA) and concanavalin A (ConA) and incubated for 5 days. IFN-γ concentration was evaluated in supernatants of stimulated blood using a commercial sandwich ELISA. Leishmania real-time PCR was also performed for assessing blood parasitemia. Dogs were treated with meglumine antimoniate and allopurinol. Sixteen dogs were classified as IFN-γ non-producers after LSA stimulation (mean ± SD: 0 ± 0 pg/mL) and 18 dogs as IFN-γ producers (mean ± SD: 2885.3 ± 4436.1 pg/mL) at the time of diagnosis (P < 0.0001). IFN-γ non-producers were classified in a more severe clinical staging than IFN-γ producers that presented a mild to moderate clinical staging (P = 0.03). In the IFN-γ non-producer group, production of IFN-γ after LSA stimulation was significantly increased during treatment especially at day 365 (P = 0.018) together with clinical improvement when compared with day 0. In contrast, IFN-γ producers maintained their IFN-γ production after LSA stimulation and no statistically significant changes were found during treatment follow-up. At diagnosis, IFN-γ non-producers showed a significantly higher blood parasitemia versus IFN-γ -producers (P = 0.005). IFN-γ non-producers drastically reduced blood parasitemia to minimum values at day 365 when compared with day 0 (P = 0.017). No significant differences were found at day 365 in blood parasitemia of IFN-γ producers compared to pre-treatment. At diagnosis, L. infantum specific antibodies were higher in IFN-γ non-producers than IFN-γ producers (P = 0.014). A marked reduction of antibody levels was found at day 365 when compared with day 0 in IFN-γ non-producers (P = 0.005) and producers (P = 0.001). These results demonstrate that IFN-γ concentration increases with long-term anti-Leishmania treatment together with clinical improvement in dogs that do not produce IFN-γ at diagnosis. Together with clinical recovery, reduction in blood parasitemia and L. infantum specific antibodies, tracking IFN-γ concentration could constitute an important prognostic tool for immune monitoring in CanL

Hepatozoon canis in three imported dogs: a new tickborne disease reaching the United Kingdom

An increasing number of non-endemic vectorborne pathogens have been described in dogs imported to the UK in the past two decades. Recently, an outbreak of canine babesiosis in south-east England has raised veterinary awareness with regard to the impact of such diseases on the UK canine population. Canine hepatozoonosis, caused by Hepatozoon canis and transmitted by the ingestion of Rhipicephalus sanguineus ticks, is widespread in the Mediterranean basin. Herein we describe the first three molecularly confirmed clinical cases of canine hepatozoonosis in dogs imported into the UK. Veterinarians in the UK should be aware of H canis as a potential infection in imported dogs, especially in the face of the expanding distribution of R sanguineus ticks in Europe

Acute febrile illness is associated with Rickettsia spp infection in dogs

BACKGROUND: Rickettsia conorii is transmitted by Rhipicephalus sanguineus ticks and causes Mediterranean Spotted Fever (MSF) in humans. Although dogs are considered the natural host of the vector, the clinical and epidemiological significance of R. conorii infection in dogs remains unclear. The aim of this prospective study was to investigate whether Rickettsia infection causes febrile illness in dogs living in areas endemic for human MSF. METHODS: Dogs from southern Italy with acute fever (n = 99) were compared with case–control dogs with normal body temperatures (n = 72). Serology and real-time PCR were performed for Rickettsia spp., Ehrlichia canis, Anaplasma phagocytophilum/A. platys and Leishmania infantum. Conventional PCR was performed for Babesia spp. and Hepatozoon spp. Acute and convalescent antibodies to R. conorii, E. canis and A. phagocytophilum were determined. RESULTS: The seroprevalence rates at first visit for R. conorii, E. canis, A. phagocytophilum and L. infantum were 44.8%, 48.5%, 37.8% and 17.6%, respectively. The seroconversion rates for R. conorii, E. canis and A. phagocytophilum were 20.7%, 14.3% and 8.8%, respectively. The molecular positive rates at first visit for Rickettsia spp., E. canis, A. phagocytophilum, A. platys, L. infantum, Babesia spp. and Hepatozoon spp. were 1.8%, 4.1%, 0%, 2.3%, 11.1%, 2.3% and 0.6%, respectively. Positive PCR for E. canis (7%), Rickettsia spp. (3%), Babesia spp. (4.0%) and Hepatozoon spp. (1.0%) were found only in febrile dogs. The DNA sequences obtained from Rickettsia and Babesia PCRs positive samples were 100% identical to the R. conorii and Babesia vogeli sequences in GenBank®, respectively. Febrile illness was statistically associated with acute and convalescent positive R. conorii antibodies, seroconversion to R. conorii, E. canis positive PCR, and positivity to any tick pathogen PCRs. Fourteen febrile dogs (31.8%) were diagnosed with Rickettsia spp. infection based on seroconversion and/or PCR while only six afebrile dogs (12.5%) seroconverted (P = 0.0248). The most common clinical findings of dogs with Rickettsia infection diagnosed by seroconversion and/or PCR were fever, myalgia, lameness, elevation of C-reactive protein, thrombocytopenia and hypoalbuminemia. CONCLUSIONS: This study demonstrates acute febrile illness associated with Rickettsia infection in dogs living in endemic areas of human MSF based on seroconversion alone or in combination with PCR

Association between canine leishmaniosis and Ehrlichia canis co-infection: a prospective case-control study

Abstract Background In the Mediterranean basin, Leishmania infantum is a major cause of disease in dogs, which are frequently co-infected with other vector-borne pathogens (VBP). However, the associations between dogs with clinical leishmaniosis (ClinL) and VBP co-infections have not been studied. We assessed the risk of VBP infections in dogs with ClinL and healthy controls. Methods We conducted a prospective case-control study of dogs with ClinL (positive qPCR and ELISA antibody for L. infantum on peripheral blood) and clinically healthy, ideally breed-, sex- and age-matched, control dogs (negative qPCR and ELISA antibody for L. infantum on peripheral blood) from Paphos, Cyprus. We obtained demographic data and all dogs underwent PCR on EDTA-blood extracted DNA for haemoplasma species, Ehrlichia/Anaplasma spp., Babesia spp., and Hepatozoon spp., with DNA sequencing to identify infecting species. We used logistic regression analysis and structural equation modelling (SEM) to evaluate the risk of VBP infections between ClinL cases and controls. Results From the 50 enrolled dogs with ClinL, DNA was detected in 24 (48%) for Hepatozoon spp., 14 (28%) for Mycoplasma haemocanis, 6 (12%) for Ehrlichia canis and 2 (4%) for Anaplasma platys. In the 92 enrolled control dogs, DNA was detected in 41 (45%) for Hepatozoon spp., 18 (20%) for M. haemocanis, 1 (1%) for E. canis and 3 (3%) for A. platys. No Babesia spp. or “Candidatus Mycoplasma haematoparvum” DNA was detected in any dog. No statistical differences were found between the ClinL and controls regarding age, sex, breed, lifestyle and use of ectoparasitic prevention. A significant association between ClinL and E. canis infection (OR = 12.4, 95% CI: 1.5–106.0, P = 0.022) was found compared to controls by multivariate logistic regression. This association was confirmed using SEM, which further identified that younger dogs were more likely to be infected with each of Hepatozoon spp. and M. haemocanis, and dogs with Hepatozoon spp. were more likely to be co-infected with M. haemocanis. Conclusions Dogs with ClinL are at a higher risk of co-infection with E. canis than clinically healthy dogs. We recommend that dogs diagnosed with ClinL should be tested for E. canis co-infection using PCR

Canine leishmaniasis: the key points for qPCR result interpretation

Background: Diagnosis and follow up of CanL is difficult since the range of clinical signs is varied and seroprevalence is high in endemic areas. The aims of this study were: i) demonstrate the advantages of Leishmania qPCR to diagnose and control CanL and highlight its prognostic value and ii) propose guidelines for tissue selection and infection monitoring. Findings: This study included 710 dogs living in an endemic area of leishmaniasis. Forty percent (285/710) exhibited clinical signs consistent with CanL. Infection was detected in 36.3% (258/710) of the dogs of which 4.5% (32/710) were detected by qPCR, 16.2% (115/710) detected by ELISA and 15.6% (111/710) tested positive for both tests. Only 17.9% (127/710) of the dogs were classified sick (affected) with CanL. All symptomatic dogs with medium or high ELISA titers were qPCR-positive in blood samples. All dogs with inconclusive or low ELISA results with high or medium qPCR parasitemia values developed the disease. Seventy one percent of asymptomatic ELISA-positive dogs confirmed by qPCR (medium to high parasitemia) developed the disease. Bone marrow or lymph node aspirate should be selected to ensure the absence of the parasite in asymptomatic dogs: 100-1,000 parasites/ml in bone marrow are detectable in blood, whereas lower parasite loads are usually negative. Almost 10% of negative samples in blood were positive in conjunctival swabs. Conclusions: Because qPCR allows parasite quantification, it is an effective tool to confirm a diagnosis of CanL in (i) cases of inconclusive ELISA results, (ii) when the dog has not yet seroconverted, or (iii) for treatment monitoring

A survey of canine tick-borne diseases in India

Background: There are few published reports on canine Babesia, Ehrlichia, Anaplasma, Hepatozoon and haemotropic Mycoplasma infections in India and most describe clinical disease in individual dogs, diagnosed by morphological observation of the microorganisms in stained blood smears. This study investigated the occurrence and distribution of canine tick-borne disease (TBD) pathogens using a combination of conventional and molecular diagnostic techniques in four cities in India. Results: On microscopy examination, only Hepatozoon gamonts were observed in twelve out of 525 (2.3%; 95% CI: 1.2, 4) blood smears. Using polymerase chain reaction (PCR), a total of 261 from 525 dogs (49.7%; 95% CI: 45.4, 54.1) in this study were infected with one or more canine tick-borne pathogen. Hepatozoon canis (30%; 95% CI: 26.0, 34.0) was the most common TBD pathogen found infecting dogs in India followed by Ehrlichia canis (20.6%; 95% CI: 17.2, 24.3), Mycoplasma haemocanis (12.2%; 95% CI: 9.5, 15.3), Anaplasma platys (6.5%; 95% CI: 4.5, 8.9), Babesia vogeli (5.5%, 95% CI: 3.7, 7.8) and Babesia gibsoni (0.2%, 95% CI: 0.01, 1.06). Concurrent infection with more than one TBD pathogen occurred in 39% of cases. Potential tick vectors, Rhipicephalus (most commonly) and/or Haemaphysalis ticks were found on 278 (53%) of dogs examined. Conclusions: At least 6 species of canine tick-borne pathogens are present in India. Hepatozoon canis was the most common pathogen and ticks belonging to the genus Rhipicephalus were encountered most frequently. Polymerase chain reaction was more sensitive in detecting circulating pathogens compared with peripheral blood smear examination. As co-infections with canine TBD pathogens were common, Indian veterinary practitioners should be cognisant that the discovery of one such pathogen raises the potential for multiple infections which may warrant different clinical management strategies