Feline leishmaniosis with focus on ocular manifestation: a case report.

BACKGROUND: In Europe, feline leishmaniosis is commonly caused by Leishmania infantum. There is little knowledge regarding pathogenesis, ocular manifestations and long-term follow-ups in cats with leishmaniosis. FINDINGS: A 6-year-old female, spayed European Shorthair cat was imported from Spain to Germany 2 years prior to its first clinical presentation. The cat showed lethargy, weight loss, ulcerative lesions on the front limbs and high-grade chronic uveitis. The diagnosis of L. infantum infection was based on the cytological finding of amastigotes in skin lesions, positive qPCR of EDTA-blood and positive PCR of a cyto-brush sample from the conjunctiva. Supportive findings included positive serology by IFAT, serum protein capillary electrophoresis with peaks in alpha2- and gamma-globulin sections and marked elevation of SAA. Enucleation had to be performed on day 288 on both eyes because of blindness, glaucoma and high-grade uveitis. Histologically, high numbers of Leishmania spp. amastigotes were found in histiocytes. IFAT and PCR were positive in the aqueous humor in both eyes, respectively. Feline leukemia virus antigen and feline immunodeficiency virus antibody testings were positive. Hematological and biochemical results revealed mild leukocytosis with lymphocytosis, monocytosis and eosinopenia as well as marked elevation of SAA and hyperglobulinemia. The cat was treated with allopurinol, responded well and was still alive at follow-up on day 288 after first presentation. However, enucleation was necessary because of refractory glaucoma and uveitis.  CONCLUSION: For the first time, ocular evidence of Leishmania IgG antibodies was demonstrated in the aqueous humor of both eyes in cats. There is limited knowledge about the pathogenesis, treatment options and outcomes in cats infected with L. infantum. This case report supports the hypothesis that immunosuppression increases the risk of clinical signs of leishmaniasis in cats. Alpha2- and gamma-globulin peaks in serum protein capillary electrophoresis are supportive criteria for the diagnosis of L. infantum infection. SAA is valuable for monitoring. Regarding ophthalmology, uveitis and glaucoma may have a poor prognosis.

[Canine leishmaniosis: an update regarding diagnostics, therapeutic approaches, and monitoring]. / Ein Update zur Leishmaniose des Hundes: Diagnostik, Therapie und Monitoring.

Canine infections with Leishmania (L.) infantum are gaining significance in Germany due to rising numbers of dogs imported from endemic countries, frequent travel and changing of climatic conditions in Central Europe. Dogs without any clinical signs suspicious for vector-borne infections imported from other countries to Germany should be tested immediately after import and 6 months later. In dogs with clinical signs suspicious for leishmaniosis, direct and indirect detection methods of the pathogen as well as hematology, biochemistry, serum protein electrophoresis and C-reactive protein are recommended. For treatment and monitoring of canine leishmaniosis, the LeishVet-guidelines are highly recommended. Different therapeutic options include first-line, second-line, and third-line drugs. For dose adjustments of allopurinol, the "step plan" should be taken in consideration. Due to climatic changes, habitats of sandflies as transmitting vectors of leishmaniosis are expanding. Next to vectorial transmission mating, transplacental infections, bite wounds, and blood transfusions were described in canine leishmaniosis. Additionally, L. infantum is a zoonotic vector-borne infectious pathogen, which is important regarding the "One-health"-aspect.

First evidence of vertical Hepatozoon canis transmission in dogs in Europe.

BACKGROUND: Hepatozoon canis is a protozoal agent that is known to be transmitted by oral uptake of H. canis-infected Rhipicephalus sanguineus sensu lato ticks in dogs. Vertical transmission of H. canis has only been described once in a study evaluating dogs from Japan. The aim of this study was to investigate the parasitological status of puppies from a bitch that had tested positive for Hepatozoon spp. prior to giving birth. FINDINGS: A 4-year-old, female, pregnant dog imported from Italy (Sardinia) to Germany showed clinical signs of lethargy and tachypnoea and tested positive for H. canis by PCR. The dog gave birth to eight puppies, one of which was stillborn and another that had to be reanimated. Haematology, buffy coat analysis and a biochemistry profile were performed for each dog. EDTA-blood of the surviving seven puppies and bone marrow, liver, spleen, amniotic fluid, and umbilical cord of the stillborn puppy was tested for the presence of Hepatozoon spp. by PCR. The mother and the seven surviving puppies tested positive for H. canis by PCR at day 62 post-partum. Gamonts were detected in all dogs by buffy coat evaluation. Haematological and biochemistry results revealed mild abnormalities. In the stillborn puppy, spleen, umbilical cord, and amniotic fluid were positive for H. canis. CONCLUSION: The results confirm that vertical transmission is a possible route of H. canis infection in dogs, demonstrated by molecular detection of the pathogen in the stillborn puppy. In the seven surviving puppies, vertical transmission was the most likely transmission route. A potential impact of the level of parasitaemia on the health of puppies, as well as its pathogenesis, should be investigated further.

An ELISA for the early diagnosis of acute canine babesiosis detecting circulating antigen of large Babesia spp.

Babesia canis is the predominant Babesia species in dogs in Europe and is responsible for a severe and fatal disease. An increase in global pet tourism and a widening of the geographic distribution of the tick vector has led to the emergence of infections in areas where previously only imported cases have been reported. Due to the potential for rapid and serious disease progression, direct parasite detection by stained blood smears and light microscopy or DNA-based methods have traditionally been used for the diagnosis of acute infections. This study describes the production of a murine monoclonal antibody ('mAb BcFIII 7/1/2') that reacts to a 65kDa corpuscular epitope present in B. canis-infected erythrocytes and can be used in an ELISA to detect circulating Babesia antigen during acute infections. The sensitivity of the ELISA was 100% (95%CI: 84.5-100) as determined using blood lysate samples from 27 dogs with acute B. canis infections. Sensitivity was reduced to 53.8% in 13 patent Babesia vogeli infections (95%CI: 26.1-79.6) based on the current test design using convalescent serum from a B. canis-infected dog. The specificity was determined to be 86.4% (95%CI: 64-96.4) using 22 samples from healthy canine blood donors. In the course of acute B. canis infections, the ELISA showed a positive result at the same time as a positive PCR result was recorded. This was 24-48h before parasites could be detected by light microscopy. Convalescent samples collected from 6 B. canis-infected dogs at least 14days post treatment resulted in negative ELISA reactions. The hyper-acute to acute phase of a B. canis infection represents an emergency situation with high mortality. To increase the chances of survival, a fast and accurate diagnosis and immediate treatment is required. The current study demonstrates the opportunity of an early and specific detection of acute infections by an AgELISA that is potentially translatable to a rapid diagnostic test design.

First report of transmission of canine leishmaniosis through bite wounds from a naturally infected dog in Germany.

BACKGROUND: Canine leishmaniosis (CanL) is an important zoonosis caused by Leishmania (L.) infantum. Transmission of L. infantum to dogs (and humans) is mainly through the bite of infected sandflies, but the parasite can also be transmitted vertically, venereally and through blood transfusions of infected donors. Additionally, the direct dog-to-dog transmission through bites or wounds is suspected. RESULTS: In December 2015, a female eight-year-old Jack-Russell-Terrier was tested positive for CanL in Germany (ELISA 74, IFAT 1:4.000). The dog had never been in an endemic area, had never received a blood transfusion and had never been used for breeding. Another female Jack-Russell-Terrier (born 2009 in Spain) was kept in the same household between 2011 and 2012. That dog was imported to Germany in 2011 and was tested positive for leishmaniosis in 2012. The Spanish-born dog had received several bite wounds, i.a. in the neck, during fights with the German-born Terrier. CONCLUSION: This may be the first report of transmission of L. infantum through bite wounds from a naturally infected dog in Germany.

Detection of Dirofilaria repens microfilariae in a dog from Portugal.

Dirofilaria repens causes subcutaneous infection in dogs and cats, and is the main agent of human dirofilariosis in Europe. Detection of D. repens is described in a dog from Portugal, a finding that simultaneously represents the first presumable case of autochthonous infection with the parasite in any animal host species in the country. A mixed D. repens/Dirofilaria immitis infection (20:1 ratio) was found, with an overall density of 1267 microfilariae per millilitre of blood. Morphological features, including morphometry, and acid phosphatase histochemical staining confirmed identity of the two filarioids. Distribution of D. repens in Portugal should be further assessed at the population level both in vertebrate hosts and in vectors. Due to the zoonotic potential of the parasite, preventive measures such as the administration of microfilaricides and insect repellents should be put into practice to protect animals and public health.

Evaluation of a rapid device for serological in-clinic diagnosis of canine angiostrongylosis.

BACKGROUND: Angiostrongylus vasorum is a potentially fatal canine nematode. Due to the high variability of clinical signs and the often chronic and subtle course of the infections, the diagnosis is particularly challenging. A rapid in-clinic assay (Angio Detect Test, IDEXX Laboratories, Westbrook, Maine, USA) for the serological detection of circulating antigen and intended for routine in-clinic diagnosis has been evaluated. METHODS: Sensitivity was calculated with sera from 39 naturally infected dogs confirmed by Baermann-Wetzel analysis, while sera of 38 experimentally infected dogs were used for follow-up analyses, of which 10 were treated with imidacloprid/moxidectin. Cross-reactivity was tested with a total of 123 samples from dogs with proven parasitic infections with Toxocara canis (n = 21), Ancylostoma caninum (n = 4), Crenosoma vulpis (n = 18), Oslerus osleri (n = 3), Eucoleus aerophilus, (n = 6), Dirofilaria immitis (n = 28), Dirofilaria repens (n = 20), Acantocheilonema reconditum (n = 10) or Dipetalonema dracunculoides (n = 10) or multiple infections (n = 3). All sera were tested with the Angio Detect Test and with an ELISA for detection of circulating antigen of A. vasorum. RESULTS: The sensitivity of the Angio Detect Test was 84.6% (95% C.I. 69.5 - 94.1%), while specificity was 100% (95% C.I. 97.6 - 100%). The sensitivity of the ELISA (94.9%, 95% C.I. 82.7 - 99.3%) was comparable with previous evaluations. In experimentally infected dogs, earliest positive results with the Angio Detect Test were observed 9 weeks post inoculation and 5 weeks later all sera were Angio Detect Test positive. After anthelmintic treatment, seropositive dogs turned negative again within 3 to 7 weeks after treatment. The evaluation of the colour intensity of the test strips confirmed the delay of approximately 3-4 weeks for antigen detection by the Angio Detect™ Test compared to the ELISA and its correlation with the time after infection. CONCLUSIONS: This study provided evidence of a good sensitivity and a very high specificity of the rapid device Angio Detect Test for detection of circulating A. vasorum antigen in dogs with suspected canine angiostrongylosis, representing a very simple and useful tool to be broadly applied in veterinary practices. The rapid detection of infected dogs is a key point for initiating an indispensable and urgent therapy.

Emergence of sandflies (Phlebotominae) in Austria, a Central European country.

The possible existence of autochthonous sandfly populations in Central Europe north of the Alps has long been excluded. However, in the past years, sandflies have been documented in Germany, Belgium, and recently, also in Austria, close to the Slovenian border. Moreover, autochthonous human Leishmania and Phlebovirus infections have been reported in Central Europe, particularly in Germany. From 2010 to 2012, sandfly trapping (740 trap nights) was performed at 53 different capture sites in Austria using battery-operated CDC miniature light traps. Sites were chosen on the basis of their climate profile in the federal states Styria, Burgenland, and Lower Austria. Sandfly specimens found were transferred to 70% ethanol for conservation. Identification was based on morphological characters of the male genitalia and the female spermathecae, respectively. Altogether, 24 specimens, 22 females and 2 males, all identified as Phlebotomus (Transphlebotomus) mascittii Grassi, 1908, were found at six different sampling sites in all three federal states investigated. The highest number of catches was made on a farm in Lower Austria. Altogether, the period of sandfly activity in Austria was shown to be much longer than presumed, the earliest capture was made on July 3rd and the latest on August 28th. Sandflies have been autochthonous in Austria in small foci probably for long, but in the course of global warming, further spreading may be expected. Although P. mascittii is only an assumed vector of Leishmania spp.-data on its experimental transmission capacity are still lacking-the wide distribution of sandflies in Austria, a country thought to be free of sandflies, further supports a potential emergence of sandflies in Central Europe. This is of medical relevance, not only with respect to the transmission of Leishmania spp. for which a reservoir is given in dogs, but also with respect to the phleboviruses.

Morphometric analyses of canine blood microfilariae isolated by the Knott's test enables Dirofilaria immitis and D. repens species-specific and Acanthocheilonema (syn. Dipetalonema) genus-specific diagnosis.

BACKGROUND: Considering the increasing importance of small animals travel medicine and the spread of filariae with zoonotic potential to non-endemic European areas, routine filarial diagnosis in dogs is becoming important. Dirofilaria immitis, D. repens, Acanthocheilonema dracunculoides and A. reconditum are the most common canine filarial nematodes presenting blood circulating microfilariae (mf) which can be differentiated to species level by the acid phosphatase activity patterns or by PCR. Available data on the size of the mf vary considerably in the literature. The aim of this study was to validate morphometric criteria for filarial identification in blood samples of dogs after concentration of mf with the modified Knott's technique. METHODS: Morphometric analysis of 10 mf from samples identified to species level by acid phosphatase activity and partially confirmed by PCR were performed with specimens from 377 dogs. RESULTS: The mean length and width of D. immitis mf from 60 dogs were 301.77 ± 6.29 µm and 6.30 ± 0.26 µm, of D. repens mf from 171 dogs 369.44 ± 10.76 µm 8.87 ± 0.58 µm, of A. dracunculoides mf from 133 dogs 259.43 ± 6.69 µm and 5.09 ± 0.47 µm and of A. reconditum mf from 13 dogs 264.83 ± 5.47 µm and 4.63 ± 0.52 µm.For a subset of 30 samples, morphometric analysis was repeated with identical results in two laboratories. Furthermore, the size of mf concentrated and fixed by the Knott's technique was shown to be stable over 105 days. CONCLUSIONS: The Knott's test enables to clearly distinguish between D. immitis, D. repens and Acanthocheilonema spp. However, due to the overlapping size ranges of A. dracunculoides and A. reconditum, biochemical or molecular methods are required to distinguish these two species.

First report of venereal and vertical transmission of canine leishmaniosis from naturally infected dogs in Germany.

BACKGROUND: Canine leishmaniosis (CanL) is a zoonotic disease caused by Leishmania (L.) infantum. It is endemic to several tropical and subtropical countries but also to the Mediterranean region. It is transmitted by phlebotomine sandflies but occasional non-vector transmissions have been reported, including vertical and horizontal transmission. FINDINGS: The authors report a case of CanL in a female boxer dog from Dusseldorf, Germany, that had never been in an endemic region. A serum sample from the bitch was tested positive for antibodies against Leishmania (IFAT 1:2,000, ELISA 72). The bitch had whelped three litters, and one puppy from the third litter was also found to be seropositive for Leishmania antibodies (IFAT 1:4,000, ELISA 78). CONCLUSIONS: Up to now, despite intensive searching, the occurrence of sandflies could not be proved in the bitch's region of origin. Thus, vertical and horizontal transmission are to be discussed as possible ways of infection. This may be the first report of venereal and vertical transmission of L. infantum in naturally infected dogs in Germany.

Combining climatic projections and dispersal ability: a method for estimating the responses of sandfly vector species to climate change.

BACKGROUND: In the Old World, sandfly species of the genus Phlebotomus are known vectors of Leishmania, Bartonella and several viruses. Recent sandfly catches and autochthonous cases of leishmaniasis hint on spreading tendencies of the vectors towards Central Europe. However, studies addressing potential future distribution of sandflies in the light of a changing European climate are missing. METHODOLOGY: Here, we modelled bioclimatic envelopes using MaxEnt for five species with proven or assumed vector competence for Leishmania infantum, which are either predominantly located in (south-) western (Phlebotomus ariasi, P. mascittii and P. perniciosus) or south-eastern Europe (P. neglectus and P. perfiliewi). The determined bioclimatic envelopes were transferred to two climate change scenarios (A1B and B1) for Central Europe (Austria, Germany and Switzerland) using data of the regional climate model COSMO-CLM. We detected the most likely way of natural dispersal ("least-cost path") for each species and hence determined the accessibility of potential future climatically suitable habitats by integrating landscape features, projected changes in climatic suitability and wind speed. RESULTS AND RELEVANCE: Results indicate that the Central European climate will become increasingly suitable especially for those vector species with a current south-western focus of distribution. In general, the highest suitability of Central Europe is projected for all species in the second half of the 21st century, except for P. perfiliewi. Nevertheless, we show that sandflies will hardly be able to occupy their climatically suitable habitats entirely, due to their limited natural dispersal ability. A northward spread of species with south-eastern focus of distribution may be constrained but not completely avoided by the Alps. Our results can be used to install specific monitoring systems to the projected risk zones of potential sandfly establishment. This is urgently needed for adaptation and coping strategies against the emerging spread of sandfly-borne diseases.

Phlebotomus (Transphlebotomus) mascittii Grassi, 1908, in Carinthia: first record of the occurrence of sandflies in Austria (Diptera: Psychodidae: Phlebotominae).

During an entomology survey in July 2009 and July 2010, 4 males and 22 females of Phlebotomus (Transphlebotomus) mascittii were caught in southeastern Carinthia. These are the first documented records of the occurrence of Phlebotominae in Austria.

Imported and travelling dogs as carriers of canine vector-borne pathogens in Germany.

BACKGROUND: With the import of pets and pets taken abroad, arthropod-borne diseases have increased in frequency in German veterinary practices. This is reflected by 4,681 dogs that have been either travelled to or relocated from endemic areas to Germany. The case history of these dogs and the laboratory findings have been compared with samples collected from 331 dogs living in an endemic area in Portugal. The various pathogens and the seroprevalences were examined to determine the occurrence of, and thus infection risk, for vector-borne pathogens in popular travel destinations. RESULTS: 4,681 dogs were examined serological for Leishmania infantum, Babesia canis and Ehrlichia canis. Buffy coats were detected for Hepatozoon canis and blood samples were examined for microfilariae via the Knott's test. The samples were sent in from animal welfare organizations or private persons via veterinary clinics. Upon individual requests, dogs were additionally examined serological for Anaplasma phagocytophilum, Borrelia burgdorferi and Rickettsia conorii. Overall B. canis was the most prevalent pathogen detected by antibody titers (23.4%), followed by L. infantum (12.2%) and E. canis (10.1%). Microfilariae were detected in 7.7% and H. canis in 2.7% of the examined dogs. In 332/1862 dogs A. phagocytophilum, in 64/212 B. burgdorferi and in 20/58 R. conorii was detected. Of the 4,681 dogs, in total 4,226 were imported to Germany from endemic areas. Eighty seven dogs joined their owners for a vacation abroad. In comparison to the laboratory data from Germany, we examined 331 dogs from Portugal. The prevalence of antibodies/pathogens we detected was: 62.8% to R. conorii, 58% to B. canis, 30.5% to A. phagocytophilum, 24.8% to E. canis, 21.1% to H. canis (via PCR), 9.1% to L. infantum and 5.3% to microfilariae. CONCLUSIONS: The examination of 4,681 dogs living in Germany showed pathogens like L. infantum that are non-endemic in Germany. Furthermore, the German data are similar in terms of multiple pathogen infection to the data recorded for dogs from Portugal. Based on these findings the importation of dogs from endemic predominantly Mediterranean regions to Germany as well as travelling with dogs to these regions carries a significant risk of acquiring an infection. Thus we would conclude that pet owners seek advice of the veterinarians prior to importing a dog from an endemic area or travel to such areas. In general, it might be advisable to have a European recording system for translocation of dogs.

Malaria transmission in non-endemic areas: case report, review of the literature and implications for public health management.

In non-endemic areas, malaria is rare and locally acquired infections, particularly with Plasmodium falciparum, are exceptional events. The diagnosis is, therefore, likely to be delayed or missed in patients without a relevant travel history. This report describes a case of falciparum malaria in Berlin, Germany, in a patient who had not been to an endemic area for more than a decade. Potential routes of vector-related and direct transmission were evaluated, particularly with regard to a possible danger to the public. A review of the literature was conducted regarding possible routes of transmission and their probability assessed. Genotyping of parasite isolates of this and another patient with malaria admitted 16 days before revealed homology between the two strains. In a local entomological survey, anopheline vectors on the hospital grounds as well as in the residential area of both patients were found. Despite intensive investigations, the mode of transmission remained obscure. In this context, possible routes of vector-borne and direct occupational/accidental transmission in a major European city are reviewed and discussed, providing information and guidance in case other similar events occur elsewhere. Examples for investigations and measures to be taken in such a situation are provided. When local malaria transmission within a large non-immune population cannot be ruled out, genotyping of parasite isolates, local entomological surveys, preparedness for secondary cases, expert consultations in a multidisciplinary team and careful information management are essential. Malaria acquired in non-endemic areas remains an unlikely, but possible event for which awareness needs to be maintained.

Laboratory testing of the insect repellents IR3535 and DEET against Phlebotomus mascittii and P. duboscqi (Diptera: Psychodidae).

The efficacy of the repellent IR3535 was tested and compared with DEET using two laboratory colonies of phlebotomine sand flies, Phlebotomus mascittii and Phlebotomus duboscqi. P. duboscqi, originating from Senegal, is a proven vector of Leishmania major. P. mascittii (Corsican origin) has never been demonstrated to be a vector of leishmaniasis, but it was reported in 2004 as an anthropophilic species in Germany associated with autochthonous visceral leishmaniasis in people and animals. IR3535 and DEET were tested at an effective concentration of 10% in ethanol on two (one male, one female) human volunteers. The mean protection time against P. duboscqi was 5.9h for both repellents, against P. mascittii 10.4 and 8.8 h for IR3535 and DEET, respectively.

[Canine leishmaniosis in Central Europe: retrospective survey and serological study of imported and travelling dogs]. / Canine Leishmaniose in Mitteleuropa: retrospektive Umfrage und serologische Untersuchung importierter und reisebegleitender Hunde.

Canine leishmaniosis is a common parasitic disease in Central Europe affecting dogs imported or returning from endemic countries around the Mediterranean basin. Through an internet discussion forum owners of dogs with suspected or proven leishmaniosis living in Central Europe (D, A, CH), were questioned about the dog's history. Additionally, serologic examinations of the dogs for anti-Leishmania antibodies (ELISA using antigen of promastigote stages) were offered to the participants. From February to October 2003, 291 dogs imported or returning from Southern Europe (Spain, Italy, Greece, Turkey, France, Malta, Portugal and others) were analysed. Serologically, 111 dogs (38%) were classified positive; 103 being imported and eight travelling dogs. The majority of seropositive dogs originated from Spain (67%). No significant correlation could be established between race, sex and age and the incidence of the disease. The clinical symptoms in the seropositive dogs varied widely and ranged from mild general symptoms to visceral manifestations with chronic renal failure. Skin disorders were found in 78% of the seropositive, symptomatic animals. In the country of origin or after import or return, 174 out of 291 dogs (60%) had been tested for the presence of anti-Leishmania antibodies by different immunofluorescence antibody tests (IFAT). Discrepancies between the ELISA and the various IFATs used were noted in 55 cases (32%), especially in cases of low IFAT titers. Most of the seropositive dogs (80%) had been treated against leishmaniosis. In 91% of these cases, Allopurinol as monotherapy or in combination with Glucantime had been used. For diagnostics and therapy, dog owners had spent an average of 1,100 euros (median 900 euros, maximum 5,800 euros).

A molecular analysis of the subgenus Transphlebotomus Artemiev, 1984 (Phlebotomus, Diptera, Psychodidae) inferred from ND4 mtDNA with new northern records of Phlebotomus mascittii Grassi, 1908.

A comparative molecular study was performed on the three species of Phlebotomine sandflies belonging to the subgenus Transphlebotomus Artemiev whose morphological differentiation is difficult. All three species have been suspected, but never proven, to be vectors of Leishmania infantum. The ND4 gene from mtDNA was sequenced from specimens of five populations of Phlebotomus mascittii Grassi from Belgium (the first records of Phlebotomine sandflies from this country), France, and Germany. Additionally, specimens from one population of Lebanese P. canaaniticus Adler and Theodor and of one population of Cypriot P. economidesi Leger, Depaquit and Ferte topotypes were examined. The results reveal the validity of the three taxa inferred from molecular variation and a molecular homogeneity shared by all of the populations of P. mascittii included into the present study. The epidemiological consequences are discussed.