Retrospektive Analyse der parasitologischen Untersuchungsergebnisse eines privaten Untersuchungslabors : Intestinale, respiratorische und vektorübertragene Parasitosen bei Hunden und Katzen in Deutschland (2004 – 2006)

Es wurden Untersuchungsbefunde von Kotproben, die einem kommerziellen tierärztlichen Untersuchungslabor von Tierärzten in den Jahren 2004–2006 zugesandt worden waren, ausgewertet. Somit konnten aktuelle Daten die Häufigkeit des Vorkommens von Parasitosen des Verdauungs- und Atmungstrakts bei Hunden und Katzen in Deutschland liefern. Bei Hunden wurden mittels ZnCl2-NaCl-Flotationsverfahren Toxocara-Eier in 4,6 %, Oozysten von Isospora spp. (Isospora canis, Isospora burrowsi/ohioensis) in 4,5 %, Hakenwurmeier in 1,3 %, Eier von Trichuris vulpis in 0,9 %, Eier von Toxascaris leonina und Capillaria spp. in je 0,6 %, Taeniiden-Eier in 0,27 %, Neospora caninumähnliche Oozysten in 0,13 %, Sarcocystis-Sporozysten in 0,06 % sowie Strongyloides-Eier in < 0,01 % aller Kotproben (n = 53.693) nachgewiesen. Giardiaoder Cryptosporidium-spezifische Koproantigene wurden mittels Kopro-ELISA (n = 53.534; n = 1.554 Proben) in 22,8 % bzw. 10 % der Proben festgestellt. Mittels Baermann-Trichterverfahren waren Crenosoma vulpis-Larven in 2,2 % und Angiostrongylus vasorum-Larven in 1 % von 509 Proben nachzuweisen. In weniger als 0,01 % aller eingesandten Kotproben wurden makroskopisch Proglottiden von Taenia spp., Mesocestoides spp. oder Diplopylidium/Joyeuxiella spp. gefunden. Bei Katzen wurden im Flotationsverfahren Toxocara-Eier in 4,8 %, Oozysten von Isospora spp. (Isospora felis, Isospora rivolta) in 3,8 %, Capillaria spp.-Eier in 0,5 %, Toxoplasma gondii-ähnliche Oozysten in 0,4 %, Taeniiden-Eier in 0,2 %, Hakenwurmeier und Sarcocystis-Sporozysten in je 0,1% sowie Toxascaris-Eier in 0,04 % der Kotproben (n = 26.491) nachgewiesen. Giardia-(n = 26.092 Proben) oder Cryptosporidium-spezifische (n = 624 Proben) Koproantigene wurden mittels Kopro- ELISA in 15,4 % bzw. 8,3 % der Proben festgestellt. Larven von Aelurostrongylus abstrusus waren mittels Baermann-Trichterverfahren in 2,6 % von 114 Kotproben zu finden. Weniger als 0,1 % aller eingesandten Kotproben enthielten Proglottiden von Taenia spp., Mesocestoides spp., Dipylidium caninum oder Diplopylidium/Joyeuxiella spp. Die mit dem indirekten Immunfluoreszenz-Antikörper-Test ermittelte Seroprävalenz von T. gondii-spezifischen IgM-Antikörpern (Titer &#8805; 1:16) und IgG-Antikörpern (Titer &#8805; 1:64) betrug 6,2 % (n = 2.616 Seren) bzw. 38,3 % (n = 3.693 Seren). Mischinfektionen mit Einzellern, Nematoden und Cestoden konnten nachgewiesen werden. Bei den Giardia-Koproantigen positiven Hunden im Alter zwischen 3 und 6 Monate besteht eine erhöhte Wahrscheinlichkeit, dass sie auch I. burrowsi/ohioensis- Oozysten ausscheiden. Bei der gleichen Altersklasse ist es eher unwahrscheinlich, dass sie eine Mischinfektion mit T. canis und Giardien bzw. I. canis aufweisen. Katzen, die Ausscheider von Giardia-Koproantigen und T. cati-Eiern und jünger als 6 Monate sind, scheiden höchst wahrscheinlich auch keine I. felis-Oozysten über den Kot aus. Bei ganz jungen Katzen (< 3 Monate) ist eine Mischinfektion mit Giardien und T. cati nicht zu erwarten. Direktnachweise von Babesia canis, Mikrofilarien und Hepatozoon canis gelangen in 2,1%, 0,2% bzw. 0,05% der Giemsa-gefärbten Blutausstriche (n = 7.923). Mikrofilarien wurden in 4,5% der mit dem Knott-Test untersuchten Proben (n = 440) gefunden. Zirkulierendes Dirofilaria immitis-Antigen war mittels ELISA in 1,4% der Proben (n = 9.381) festzustellen. Mittels PCR wurden B. canis-DNA in 3,3% der Blutproben (n = 15.555) und Leishmania infantum-DNA in 11 % der eingesandten Proben (n = 301) nachgewiesen. Antikörper gegen L. infantum oder B. canis wurden in 23,5% bzw. 11,5% der mittels IFAT untersuchten Serumproben (n = 23.665 bzw. 2.653) festgestellt. Leishmanien-Antikörper wurden bei den aus dem Ausland stammenden Hunden (80,6 %) und bei 4 einheimischen Hunden ohne Reiseanamnese nachgewiesen. Im Blutausstrich konnten Babesien-Merozoiten häufiger bei den aus Deutschland stammenden Tieren als bei den in anderen europäischen Ländern geborenen Hunden diagnostiziert werden. Alle auf Babesien-DNA untersuchte Hunde (n=15) aus Deutschland ohne Auslandsaufenthalt waren positiv.Faecal samples from 53.693 dogs and 26.491 cats, routinely submitted to a private veterinary laboratory between 2004 and 2006 were examined using a ZnCl2-NaCl flotation method. Toxocara canis eggs were detected in 4,6 % of canine samples, followed concerning the frequency of occurrence by Isospora spp. oocysts (Isospora canis, Isospora burrowsi/ohioensis; 4,5 % ), hookworm eggs (1,3 %), Trichuris vulpis eggs (0,9 %), Toxascaris leonina and Capillaria spp. eggs (0,6 %), taeniid eggs (0,27 %), Neospora caninum-like oocysts (0,13 %), Sarcocystis spp. sporocysts (0,06 %) and Strongyloides spp. eggs (< 0,01 %). Giardia (n = 53.534) and Cryptosporidium (n = 1.554) coproantigens were detected by ELISA in 22,8 % and 10 % of canine samples, respectively. Crenosoma vulpis larvae were detected, using the Baermann technique, in 2,2 % and Angiostrongylus vasorum larvae in 1 % of a total of 509 samples, respectively. Tapeworms (Taenia spp., Mesocestoides spp. and Diplopylidium/Joyeuxiella spp.) were detected in < 0,01 % of the faecal samples . The following gastrointestinal parasites were recorded in feline faecal samples (prevalences in brackets): Toxocara cati (4,8 %), Isospora spp. (Isospora felis, Isospora rivolta; 3,8 %), Capillaria spp. (0,5 %), Toxoplasma gondii-like oocysts (0,4 %), taeniids (0,2 %), hookworms, Sarcocystis spp. (0,1%), and Toxascaris leonina (0,04 %). Giardia (n = 26.092 samples) and Cryptosporidium (n = 624 samples) coproantigens were detected in 15,4 % and 8,3 % feline samples, respectively, using ELISA. The prevalence of Aelurostrongylus abstrusus was 2,6 % in a total of 114 faecal samples detected by Baermann technique. The segments of Taenia spp., Mesocestoides spp., Dipylidium caninum and Diplopylidium/Joyeuxiella spp. were found in < 0,1 % of feline samples. IgM (&#8805; 1:16) and IgG (&#8805; 1:64) antibodies against T. gondii were found in 6,2 % of 2.616 sera and in 38,3 % of 3.693 sera, respectively, tested by indirect fluorescent antibody test (IFAT), respectively. 208 SUMMARY Multiple parasitisation by protozoan and helminths in canine and feline faecal samples was detected. Young dogs (3 – 6 months) positive for Giardia coproantigens were at increased risk to shed also I. burrowsi/ohioensis oocysts, compared to Giardia negative puppies. Kittens positive for Giardia coproantigen younger than 6 months are not at increased risk to shed also I. felis oocysts when compared to Giardia negative kittens. Very young cats (< 3 months) are also not at increased risk to be infected with multiple parasite species (Giardia and T. cati). Blood samples were taken from dogs with a history of importation from or travelling to countries in the south or south-east of Europe, or showing clinical signs related to some vector-borne diseases. The screening of Giemsa-stained smears of 7.923 blood samples resulted in detection of Babesia canis (2,1%), microfilariae (0,2%) and Hepatozoon canis (0,05%). Knott’s Test for microfilariae was positive in 4,5% of 440 dogs. Heartworm antigen was detected by ELISA in 1,4% of 9.381 samples. Of the 15.555 and 301 blood samples analysed, 3,3 % were positive for B. canis DNA and 11 % were positive for Leishmania infantum by PCR. Antibodies against L. infantum DNA and B. canis were detected in 23,5% (n = 23.665) and 11,5% (n = 2.653) of blood samples by IFAT, respectively. Antibodies against L. infantum were detected in 80,6 % of dogs imported from endemic areas and in 4 German dogs without travel history. Babesia trophozoites were found more frequently in Giemsa-stained smears from dogs born in Germany when compared to blood samples of dogs originating from south or south-east European countries. A total of 15 blood samples of German dogs which have never been abroad, was positive for Babesia DNA

Diagnosis of gastrointestinal parasites in reptiles: comparison of two coprological methods

BACKGROUND: Exotic reptiles have become increasingly common domestic pets worldwide and are well known to be carriers of different parasites including some with zoonotic potential. The need of accurate diagnosis of gastrointestinal endoparasite infections in domestic reptiles is therefore essential, not only for the well-being of captive reptiles but also for the owners. Here, two different approaches for the detection of parasite stages in reptile faeces were compared: a combination of native and iodine stained direct smears together with a flotation technique (CNF) versus the standard SAF-method. RESULTS: A total of 59 different reptile faeces (20 lizards, 22 snakes, 17 tortoises) were coprologically analyzed by the two methods for the presence of endoparasites. Analyzed reptile faecal samples contained a broad spectrum of parasites (total occurence 93.2%, n=55) including different species of nematodes (55.9%, n=33), trematodes (15.3%, n=9), pentastomids (3.4%, n=2) and protozoans (47.5%, n=28). Associations between the performances of both methods to detect selected single parasite stages or groups of such were evaluated by Fishers exact test and marginal homogeneity was tested by the McNemar test. In 88.1% of all examined samples (n=52, 95% confidence interval [CI]=77.1 - 95.1%) the two diagnostic methods rendered differing results, and the McNemar test for paired observations showed highly significant differences of the detection frequency (P<0.0001). CONCLUSION: The combination of direct smears/flotation proved superior in the detection of flagellates trophozoites, coccidian oocysts and nematode eggs, especially those of oxyurids. SAF-technique was superior in detecting larval stages and trematode eggs, but this advantage failed to be statistically significant (P=0.13). Therefore, CNF is the recommended method for routine faecal examination of captive reptiles while the SAF-technique is advisable as additional measure particularly for wild caught animals and individuals which are to be introduced into captive collections

Serological and faecal detection of Angiostrongylus vasorum in dogs from Austria

Canine angiostrongylosis is a potentially lethal parasitic disease that can manifest itself with a broad spectrum of clinical signs, including respiratory distress, neurological and bleeding disorders, or non-specific signs. The occurrence of Angiostrongylus vasorum is widely reported in Europe, but very little is known about its presence in Austria. In this first large-scale survey, 1279 sera were collected from Austrian dogs and tested by an ELISA for the detection of circulating antigen of A. vasorum (sensitivity: 95.7%, specificity 94.0%) and by a separate ELISA detecting specific antibodies (sensitivity 81.0%, specificity 98.8%). Furthermore, 1040 faecal samples were tested for the presence of lungworm first stage larvae (L1). One dog (0.1%, 95% confidence intervals [CI]: 0.0-0.4%) was positive in both ELISAs, while 1.2% (n = 15, CI: 0.7-1.9%) of the tested dogs were antigen-positive and 1.5% (n = 19, CI: 0.9-2.3%) were positive for specific antibodies. Overall, 13 dogs (1.3%; CI: 0.7-2.1%) were positive for A. vasorum L1 while 31 dogs were positive for Crenosoma vulpis L1 (3.0%; CI: 2.0-4.2%). One dog shed L1 from both A. vasorum and C. vulpis (0.1%, CI: 0.0-0.5%). Dogs positive for A. vasorum originated from northeast, southeast and south Austria (antigen and/or antibody detection), but also from north, west and southwest Austria (antibody detection) and from northeast and west Austria (L1 detection). One of 88 blood samples (1.1%, CI: 0.0-6.2%) submitted from the eastern part of Austria was positive by a rapid assay for A. vasorum antigen detection (Angio Detect™). Crenosoma vulpis positive samples originated from northwest, north, northeast, south and west Austria. These results confirm the very sporadic occurrence of A. vasorum in the investigated areas of the country. However, due to the substantial infectious pressure from the surrounding countries and the free circulation of dogs and foxes acting as wildlife reservoirs and due to clinical relevance for infected dogs, it is crucial to maintain disease awareness also in areas where the parasite has not yet been detected

Current surveys of the seroprevalence of Borrelia burgdorferi, Ehrlichia canis, Anaplasma phagocytophilum, Leishmania infantum, Babesia canis, Angiostrongylus vasorum and Dirofilaria immitis in dogs in Bulgaria

Canine vector-borne diseases (CVBDs) have increasingly become a focus of interest in recent years. Some of the CVBDs are zoonotic and may therefore also represent a risk for the human population. Different factors are in discussion to explain the expansion of vectors and pathogens into formerly unaffected areas. Knowledge of the prevalence and distribution of CVBDs in Bulgaria is scant overall and most data rely on single case descriptions. The aim of the present study was to determine the seroprevalence of important CVBDs in 167 dogs from central-southern Bulgaria (Stara Zagora), with special emphasis on hitherto uninvestigated babesiosis and angiostrongylosis, on poorly investigated Lyme borreliosis and canine granulocytic anaplasmosis, and on the potentially zoonotic dirofilariosis and leishmaniosis. Relatively high prevalence rates were documented for anti-Babesia canis antibodies, Dirofilaria immitis antigen (16.2 %; 27/167 each), anti-Ehrlichia canis (21 %; 35/167) and anti-Anaplasma phagocytophilum antibodies (30.5 - 46.1 %; 51 - 77/167), while Borrelia burgdorferi seroprevalence was low (2.4 %; 4/167). All samples were negative for Leishmania infantum antibodies and Angiostrongylus vasorum antigen and antibodies. In total, 64.7 % (108/167) of the samples indicated infection or exposure to at least one agent and a high proportion of dual infections (39.8 %; 43/108) was demonstrated. Multiple infections with up to four different organisms were also detected. Our data underline the importance of CVBDs and especially of co-infections which could influence the clinical outcome in dogs

Seroprevalence and current infections of canine vector-borne diseases in Costa Rica

Domestic dogs may carry several vector-borne pathogens, including zoonotic agents, especially in tropical regions like Central America. The epidemiology of these pathogens is prone to change due to urbanization, trade and travel as well as climate change, necessitating repeated monitoring. This study aims to present a comprehensive picture of canine vector-borne diseases in Costa Rica, combining data on seroprevalence with molecular species identification of the causative pathogens. In this survey, 294 dogs from all seven provinces of Costa Rica were included. After a clinical examination, diagnostic blood samples were analyzed with regard to packed cell volume (PCV) and presence of microfilaria. Serum samples were tested for antibodies against Ehrlichia spp., Anaplasma spp., Babesia spp., Borrelia burgdorferi sensu lato (s.l.) as well as antigen of Dirofilaria immitis. Seropositive and microfilaremic blood samples were analyzed by PCR to detect current infections and identify the pathogen species. Overall, 45.24% (133/294, 95% CI: 39.45–51.11%) of dogs were seropositive for at least one of the tested pathogens. Seroprevalence was highest for Ehrlichia spp. (39.46%, 116/294, 95% CI: 33.83–45.29%), followed by Babesia spp. (23.13%, 68/294, 95% CI: 18.43–28.38%), Anaplasma spp. (13.27%, 39/294, 95% CI: 9.61–17.69%), and B. burgdorferi s.l. (0.34%, 1/294, 95% CI: 0.01–1.88%). Generalized linear mixed models indicated a significant association of Ehrlichia-, Anaplasma- and Babesiaseropositivity, as well as a significant effect of age and breed on Ehrlichia-seropositivity. Furthermore, a statistically significant negative effect of Ehrlichia-, Anaplasma-, and Babesia-seropositivity on PCV was found. Regarding current infections, Ehrlichia canis DNA was detected in 51.72% (60/116, 95% CI: 42.26–61.10%) of Ehrlichia-seropositive dogs, while Ehrlichia ewingii and Ehrlichia chaffeensis were not detected. Furthermore, 10.26% (4/39, 95% CI: 2.87–24.22%) of Anaplasma-seropositive dogs were coinfected with Anaplasma phagocytophilum and Anaplasma platys, while one animal (2.56%, 95% CI: 0.65–13.48%) was infected with A. phagocytophilum only. Among Babesiaseropositive dogs, Babesia vogeli and Hepatozoon canis were detected in one animal each (1.47%, 1/68, 95% CI: 0.04–7.92%). Dirofilaria immitis antigen was detected in 4.42% (13/294, 95% CI: 2.38–7.44%) of dogs. In microfilaremic animals, D. immitis as well as Acanthocheilonema reconditum infections were identified. This survey demonstrates that canine vector-borne pathogens, including zoonotic agents like A. phagocytophilum and D. immitis, are widespread in Costa Rica. Thus, protection of dogs from disease-transmitting vectors is recommended from an animal welfare as well as public health perspective.Los perros domésticos pueden ser portadores de varios patógenos transmitidos por vectores, incluidos agentes zoonóticos, especialmente en regiones tropicales como América Central. La epidemiología de estos patógenos es propensa a cambiar debido a la urbanización, el comercio y los viajes, así como al cambio climático, lo que hace necesario un seguimiento repetido. Este estudio pretende presentar un panorama completo de las enfermedades caninas transmitidas por vectores en Costa Rica, combinando datos de seroprevalencia con la identificación molecular de especies de los patógenos causantes. En este estudio se incluyeron 294 perros de las siete provincias de Costa Rica. Tras un examen clínico, se analizaron muestras de sangre para el diagnóstico en relación con el volumen celular empaquetado (PCV) y la presencia de microfilarias. Las muestras de suero se analizaron para detectar anticuerpos contra Ehrlichia spp., Anaplasma spp., Babesia spp. y Borrelia burgdorferi sensu lato (s.l.), así como el antígeno de Dirofilaria immitis. Las muestras de sangre seropositivas y microfilarémicas se analizaron mediante PCR para detectar las infecciones actuales e identificar la especie patógena. En general, el 45,24% (133/294, IC 95%: 39,45-51,11%) de los perros eran seropositivos para al menos uno de los patógenos analizados. La seroprevalencia fue mayor para Ehrlichia spp. (39,46%, 116/294, IC 95%: 33,83-45,29%), seguido de Babesia spp. (23,13%, 68/294, IC 95%: 18,43-28,38%), Anaplasma spp. (13,27%, 39/294, IC 95%: 9,61-17,69%), y B. burgdorferi s.l. (0,34%, 1/294, IC 95%: 0,01-1,88%). Los modelos lineales mixtos generalizados indicaron una asociación significativa de Ehrlichia-, Anaplasma- y Babesiaseropositividad, así como un efecto significativo de la edad y la raza sobre la Ehrlichia-seropositividad. Además, se encontró un efecto negativo estadísticamente significativo de la seropositividad a Ehrlichia, Anaplasma y Babesia sobre el PCV. En cuanto a las infecciones actuales, se detectó ADN de Ehrlichia canis en el 51,72% (60/116, IC del 95%: 42,26-61,10%) de los perros seropositivos a Ehrlichia, mientras que no se detectaron Ehrlichia ewingii y Ehrlichia chaffeensis. Además, el 10,26% (4/39, IC 95%: 2,87-24,22%) de los perros seropositivos a Anaplasma estaban coinfectados con Anaplasma phagocytophilum y Anaplasma platys, mientras que un animal (2,56%, IC 95%: 0,65-13,48%) estaba infectado sólo con A. phagocytophilum. Entre los perros positivos a Babesias, se detectaron Babesia vogeli y Hepatozoon canis en un animal cada uno (1,47%, 1/68, IC 95%: 0,04-7,92%). El antígeno de Dirofilaria immitis se detectó en el 4,42% (13/294, IC 95%: 2,38-7,44%) de los perros. En los animales microfilarémicos se identificaron infecciones por D. immitis así como por Acanthocheilonema reconditum. Este estudio demuestra que los patógenos caninos transmitidos por vectores, incluidos los agentes zoonóticos como A. phagocytophilum y D. immitis, están muy extendidos en Costa Rica. Así pues, se recomienda proteger a los perros de los vectores transmisores de enfermedades, tanto desde el punto de vista del bienestar animal como de la salud pública.University of Veterinary Medicine Hannover, AlemaniaUniversidad Nacional, Costa RicaIDEXX Laboratories, AlemaniaEscuela de Medicina Veterinari

Molecular identification of Sarcocystis spp. helped to define the origin of green pythons (Morelia viridis) confiscated in Germany.

Sarcocystis spp. represent apicomplexan parasites. They usually have a heteroxenous life cycle. Around 200 species have been described, affecting a wide range of animals worldwide, including reptiles. In recent years, large numbers of reptiles have been imported into Europe as pets and, as a consequence, animal welfare and species protection issues emerged. A sample of pooled feces from four confiscated green pythons (Morelia viridis) containing Sarcocystis spp. sporocysts wasinvestigated. These snakes were imported for the pet trade and declared as being captive-bred. Full length 18S rRNA genes were amplified, cloned into plasmids and sequenced. Two different Sarcocystis spp. sequences were identified and registered as Sarcocystis sp. from M. viridis in GenBank. Both showed a 95?97% sequence identity with the 18S rRNA gene of Sarcocystis singaporensis. Phylogenetic analysis positioned these sequences together with other Sarcocystis spp. from snakesand rodents as definitive and intermediate hosts (IH), respectively. Sequence data and also the results of clinical and parasitological examinations suggest that the snakeswere definitive hosts for Sarcocystis spp. that circulate in wild IH. Thus, it seems unlikely that the infected snakes had been legally bred. Our research shows that information on the infection of snakes with Sarcocystis spp. may be used to assess compliance with regulations on the trade with wildlife species.Fil: Moré, Gastón Andrés. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institut; Alemania. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Pantchev, Nikola. IDEXX Vet Med Lab; AlemaniaFil: Herrmann, Daland C.. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institut; AlemaniaFil: Globokar Vrhovec, Majda. IDEXX Vet Med Lab; AlemaniaFil: Öfner, Sabine. Reptile Rescue Centre Munich; AlemaniaFil: Conraths, Franz J.. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institut; AlemaniaFil: Schares, Gereon. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institut; Alemani

First Cases of Natural Infections with Borrelia hispanica in Two Dogs and a Cat from Europe

Canine cases of relapsing fever (RF) borreliosis have been described in Israel and the USA, where two RF species, Borrelia turicatae and Borrelia hermsii, can cause similar clinical signs to the Borrelia persica in dogs and cats reported from Israel, including fever, lethargy, anorexia, thrombocytopenia, and spirochetemia. In this report, we describe the first clinical cases of two dogs and a cat from Spain (Cordoba, Valencia, and Seville) caused by the RF species Borrelia hispanica. Spirochetes were present in the blood smears of all three animals, and clinical signs included lethargy, pale mucosa, anorexia, cachexia, or mild abdominal respiration. Laboratory findings, like thrombocytopenia in both dogs, may have been caused by co-infecting pathogens (i.e., Babesia vogeli, confirmed in one dog). Anemia was noticed in one of the dogs and in the cat. Borrelia hispanica was confirmed as an infecting agent by molecular analysis of the 16S rRNA locus. Molecular analysis of housekeeping genes and phylogenetic analyses, as well as successful in vitro culture of the feline isolate confirmed the causative agent as B. hispanica

A real-time quantitative polymerase chain reaction for the specific detection of Hammondia hammondi and its differentiation from Toxoplasma gondii

INTRODUCTION: Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites, but only T. gondii is zoonotic. Both species use felids as definitive hosts and cannot be differentiated by oocyst morphology. In T. gondii, a 529-base pair (bp) repetitive element (TgREP-529) is of utmost diagnostic importance for polymerase chain reaction (PCR) diagnostic tests. We identified a similar repetitive region in the H. hammondi genome (HhamREP-529). METHODS: Based on reported sequences, primers and probes were selected in silico and optimal primer probe combinations were explored, also by including previously published primers. The analytical sensitivity was tested using serial dilutions of oocyst DNA. For testing analytical specificity, DNA isolated from several related species was used as controls. The newly established TaqMan PCR (Hham-qPCR1) was applied to tissues collected from H. hammondi-infected gamma-interferon gene knockout (GKO) mice at varying time points post-infection. RESULTS: Ten forward and six reverse primers were tested in varying combinations. Four potentially suitable dual-labelled probes were selected. One set based on the primer pair (Hham275F, Hham81R) and the probe (Hham222P) yielded optimal results. In addition to excellent analytic specificity, the assay revealed an analytical sensitivity of genome equivalents of less than one oocyst. Investigation of the tissue distribution in GKO mice revealed the presence of parasite DNA in all examined organs, but to a varying extent, suggesting 100- to 10,000-fold differences in parasitic loads between tissues in the chronic state of infection, 42 days post-infection. DISCUSSION: The use of the 529-bp repeat of H. hammondi is suitable for establishing a quantitative real-time PCR assay, because this repeat probably exists about 200 times in the genome of a single organism, like its counterpart in T. gondii. Although there were enough sequence data available, only a few of the primers predicted in silico revealed sufficient amplification; the identification of a suitable probe was also difficult. This is in accord with our previous observations on considerable variability in the 529-bp repetitive element of H. hammondi. CONCLUSIONS: The H. hammondi real-time PCR represents an important novel diagnostic tool for epidemiological and cell biological studies on H. hammondi and related parasites

Case Report of a Fatal Babesia vulpes Infection in a Splenectomised Dog

Babesia vulpes is a small Babesia prevalent in foxes in Europe and mainly clinically affects dogs in north-western Spain. A dog imported from this region that had been living in Germany for three years developed splenic torsion. After splenectomy, the dog underwent immunosuppressive therapy because of autoimmune disease due to haemotrophic Mycoplasma sp. infection. As clinical signs worsened, small Babesia were detected in a blood smear and identified as B. vulpes by molecular analysis. Anaemia, thrombocytosis, elevated liver enzymes, and renal parameters were the most significant findings in blood analysis. The dog was treated with a combination of atovaquone (20 mg/kg BW, BID), proguanil hydrochloride (8 mg/kg BW, BID) and azithromycin (10 mg/kg BW, SID), which led to an increase in the cycle threshold in real-time PCR and the absence of B. vulpes in the blood smear. However, after clinical signs deteriorated, the dog was euthanised. This case report supports the recommendation to screen imported dogs for pathogens and highlights the impact of splenectomy on the course of infection.Babesia vulpes is a small Babesia prevalent in foxes in Europe and mainly clinically affects dogs in north-western Spain. A dog imported from this region that had been living in Germany for three years developed splenic torsion. After splenectomy, the dog underwent immunosuppressive therapy because of autoimmune disease due to haemotrophic Mycoplasma sp. infection. As clinical signs worsened, small Babesia were detected in a blood smear and identified as B. vulpes by molecular analysis. Anaemia, thrombocytosis, elevated liver enzymes, and renal parameters were the most significant findings in blood analysis. The dog was treated with a combination of atovaquone (20 mg/kg BW, BID), proguanil hydrochloride (8 mg/kg BW, BID) and azithromycin (10 mg/kg BW, SID), which led to an increase in the cycle threshold in real-time PCR and the absence of B. vulpes in the blood smear. However, after clinical signs deteriorated, the dog was euthanised. This case report supports the recommendation to screen imported dogs for pathogens and highlights the impact of splenectomy on the course of infection