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.

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.

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.

Phlebotomine sand flies in Southwest Germany: an update with records in new locations.

BACKGROUND: Vector-borne diseases (VBD) are of growing global importance. Sand flies are potential vectors for phleboviruses (family Phenuiviridae) including Toscana virus (TOSV), Sicilian virus, Sandfly fever, Naples virus, and Leishmania parasites in Europe. To date, only two phlebotomine species have been recorded for Germany: Phlebotomus perniciosus and Phlebotomus mascittii. This study updates the distribution and abundance of the two occurring species. METHODS: An entomological field study was carried out during 2015-2018 to assess the abundance of sand flies in Southwest Germany within the federal states Baden-Wuerttemberg (BW) and Rhineland-Palatinate (RLP). A total of 176 collection sites were examined using CDC light traps. RESULTS: A total of 149 individuals of P. mascittii were collected. During 2015-2018, P. mascittii was found at all sites known positive from previous studies and was detected at 15 additional sites previously unknown for the presence of sand flies. Although the environment has changed considerably in 30 years, no significant difference in sand fly dynamics and distribution was found. Phlebotomus perniciosus has only been trapped once since 2001. CONCLUSIONS: This study showed that sand flies occur in different areas in Southern Germany where they had not been recorded previously. Therefore, it can be assumed that they are more widespread than expected. In addition, sand flies could be found over several years at the same trapping sites, indicating population stability. This supports the need for continued surveillance of possible vector populations and urgent clarification of the vector competence of P. mascittii.

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.

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.

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.

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.

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.