Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

[Dog and fox faecal contamination of farmland]. / Kontamination landwirtschaftlicher Nutzflächen durch Hunde- und Fuchskot.

The contamination with faeces from dogs and foxes was documented on 14 different grassland areas in the canton of Zurich, Switzerland, over one year. A total of 402 dog and 58 fox faecal samples were collected from the grasslands, further 236 faecal samples were retrieved from Robidog® units (disposal units for dog waste bags) in the immediate vicinity. The degree of fecal contamination per 100 m2 and year was 0.07-0.75 for dog samples and 0-0.06 for fox samples. Dog faeces from Robidog® units and grasslands contained stages of the following parasites, respectively (sedimentation/flotation method): Toxocara sp. (2.5%; 1.2%), Taenia crassiceps (with molecular confirmation; 0.8%; 0.2%), Capillaria sp. (0.4%; 0.7%), Trichuris sp. (0.8%; 1%), Isospora sp. (2.1%; 2%) and Angiostrongylus vasorum (0.4%; 0.5%). In fox faeces parasite stages were more frequently detected: 19% Toxocara sp., 8.6% Taenia crassiceps, 6.9% Echinococcus multilocularis, 60.3% Capillaria sp., 29.3% Trichuris sp. In two fecal samples from foxes, Taenia saginata eggs or Toxoplasmagondii oocysts were confirmed by molecular analyses, these findings may be explained as an intestinal passage after coprophagy of human or cat feces, respectively. Therefore, foxes can also indirectly play a role in parasite transmission to livestock.

Clinical, laboratory and pathological findings in cats experimentally infected with Aelurostrongylus abstrusus.

Aelurostrongylus abstrusus parasitizes the respiratory tract and can heavily affect the breathing and general condition of cats. Experimental infections of six cats were initiated by intragastric administration with 100 or 800 third-stage larvae (L3) obtained from the terrestrial snail Helix aspersa. First-stage larvae were isolated from faecal samples after 35-41 days post infection (dpi) in five animals and until end of study (84 dpi) in two cats. Cough and respiratory sounds were observed starting from 28 to 41 dpi and dyspnoea and panting starting from 52 dpi. All cats had enlarged lymph nodes and, starting from 56 dpi, reduced body weight, and four cats showed intermittent reduced general condition with apathia and anorexia. Eosinophilia and leucocytosis partially with massive lymphocytosis, and occasional basophilia and monocytosis were observed. Mild anaemia was present in five cats, while alterations in coagulation parameters suggested stimulation of the coagulation cascade with increased consumption of coagulation factors (delayed PT, hypofibrinogenemia). Adult A. abstrusus specimens were isolated from the five patent cats at necropsy and all six cats showed pathological changes in the lungs, including disseminated inflammatory cell infiltrates, often associated with incorporated larvae and eggs. There was some degree of overlap between the severity and the inoculation doses. Infections starting from 100 L3 of A. abstrusus had an impact on the lung tissues and on the health of the cats, despite the presence of only mild haematological abnormalities. Due to the worldwide occurrence of feline lung worms, parasitic infections should be considered in the differential diagnosis of lung diseases regardless of the presence of clinical signs and larval excretion.

Trypanosomiasis: An emerging disease in Alpine swift (Tachymarptis melba) nestlings in Switzerland?

Alpine swifts (Tachymarptis melba) are sub-Saharan migratory birds, which, in Switzerland, nest in colonies that have been continuously monitored for over 40 years. In the summer of 2022, despite favourable environmental conditions, an unexpectedly high number of sudden mortalities (30-80%) occurred in 20 to 45-day-old nestlings from several nesting sites, of which 3 were monitored in detail. Nestlings submitted for post-mortem analysis (n = 5) were in good body condition but exhibited extensive subcutaneous haematomas (n = 5), myocardial petechiae (n = 2) and stunted growth of primary feathers (n = 1). In all birds, 4-5 µm large, amastigote-like protozoans were identified in skeletal and cardiac muscle sections. These tissues tested positive in a PCR targeting the 18S-rRNA gene of Trypanosoma spp. Amplified sequences showed 99.63% identity with sequences of Trypanosoma corvi (JN006854 and AY461665) and Trypanosoma sp. (AJ620557, JN006841). 72 blood smears of 45-day-old nestlings from two colonies were assessed, of which 20 contained trypomastigote forms, some with high parasitaemia (highest average of 56.4 in 10 high power fields, 400x magnification). Trypomastigote morphometrics (n = 36; mean total length = 30.0 µm; length of free flagellum = 5.8 µm) were consistent with those of T. bouffardi. These findings suggest that an avian trypanosomiasis causing mass nestling mortality could be an emerging disease in Swiss Alpine swift populations.

Neospora caninum NC-6 Argentina induces fetopathy in both serologically positive and negative experimentally inoculated pregnant dams.

Neospora caninum infection is a major cause of abortion in cattle. The objectives of this study were to genetically characterize the N. caninum NC-6 Argentina isolate using a multilocus microsatellite analysis approach and to study its biological behavior by experimental inoculations into seronegative and seropositive pregnant cattle, evaluating the humoral and cellular immune response elicited and the occurrence of transplacental transmission and fetopathy. Pregnant cows (65 days of gestation) seropositive and seronegative to N. caninum were intravenously inoculated with tachyzoites of the NC-6 Argentina N. caninum strain and slaughtered at 108 ± 2 days of gestation. Serum samples were analyzed for N. caninum antibodies by indirect fluorescent antibody test. The cellular immune response was analyzed by detection of gamma interferon (γIFN) production in blood cells. Tissue samples from dams, fetuses, and placental cotyledons were processed by histopathological and immunohistochemical techniques and examined for N. caninum DNA by PCR. Positive DNA samples were further analyzed by multilocus microsatellite typing for N. caninum. Inoculated animals had significantly higher N. caninum antibody titers and γIFN production than control animals. One seropositive inoculated cow aborted, one seronegative cow had a non-viable fetus, and the remaining fetuses from the experimentally inoculated dams had histopathologic lesions. The PCR was positive in 3/4 fetuses from seronegative inoculated cows and in 2/3 fetuses from seropositive inoculated cows. Multilocus microsatellite analysis revealed that the N. caninum DNA present in fetuses and placentas had an identical pattern to NC-6 Argentina strain. The NC-6 Argentina strain proved to be able to cross the placenta and to induce fetopathy in both the seropositive and seronegative dams.

Zoonotic potential of guinea pigs: Outbreak of cryptosporidiosis combined with chlamydiosis in a breeding guinea pig herd.

INTRODUCTION: In a guinea pig herd with 26 breeding animals, several individuals of all age categories died (16/26) after three animals had been newly introduced from another herd. Furthermore, the population suffered of apathy, anorexia, severe weight loss and conjunctivitis, as well as abortions and stillbirths. At the same time, the owner experienced a SARS-CoV-2 infection with pneumonia, which was confirmed by taking a PCR test. Chlamydia caviae was detected from the conjunctiva and vagina/uterus in one juvenile animal together with an intestinal Cryptosporidium wrairi infection. Oocysts were found histologically in the small intestine, which was confirmed by PCR. C. wairi is a parasite adapted to guinea pigs with zoonotic potential, which causes diarrhoea with frequent deaths in larger guinea pig herds. C. caviae is also a zoonotic pathogen and often the cause of conjunctivitis, pneumonia and abortions in guinea pigs and can lead to upper respiratory tract disease, conjunctivitis but also severe pneumonia in humans. The increased death cases and the clinical signs could be traced back to an infection with Cryptosporidium wrairi, complicated by a co-infection of C. caviae. We suspect that the abortions were caused by C. caviae, but since the population was treated with various antibiotics effective against chlamydial infections, it was no longer possible to verify this by PCR testing. Unfortunately, more animals succumbed and finally only two animals of the originally 26 were left. With this case report, we would like to point out to veterinarians that guinea pigs can be an important source of zoonotic infections for various pathogens, especially since they are popular pets and often come into close contact with children where hygiene might not always be strictly followed.

INTRODUCTION: Dans un groupe de cobayes de 26 animaux reproducteurs, plusieurs individus de toutes les catégories d'âge sont morts (16/26) après l'introduction de trois animaux provenant d'un autre groupe. En outre, la population a souffert d'apathie, d'anorexie, de perte de poids sévère et de conjonctivite ainsi que d'avortements et de mortinatalité. La présence de Chlamydia caviae a pu être détectée dans la conjonctive et le vagin/utérus d'un animal juvénile, ainsi qu'une infection intestinale à Cryptosporidium wrairi. Des oocystes ont été trouvés histologiquement dans l'intestin grêle, ce qui a été confirmé par PCR. C. wairi est un parasite adapté aux cobayes avec un potentiel zoonotique, qui provoque des diarrhées avec des morts fréquentes dans les grands groupes de cobayes. C. caviae est également un agent pathogène zoonotique et est souvent à l'origine de conjonctivites, de pneumonies et d'avortements chez les cobayes ; il peut entraîner des maladies des voies respiratoires supérieures, des conjonctivites mais aussi des pneumonies graves chez l'homme. L'augmentation des cas de décès et les signes cliniques pourraient être attribués à une infection par Cryptosporidium wrairi, compliquée par une co-infection par C. caviae. Nous soupçonnons que les avortements ont été causés par C. caviae, mais comme la population a été traitée avec divers antibiotiques efficaces contre les infections à chlamydia, il n'était plus possible de le vérifier par des tests PCR. Malheureusement, d'autres animaux ont succombé et il ne restait finalement que deux animaux sur les 26 d'origine. Avec ce rapport de cas, nous aimerions attirer l'attention des vétérinaires sur le fait que les cochons d'Inde peuvent être une source importante d'infections zoonotiques pour divers pathogènes, d'autant plus qu'il s'agit d'animaux de compagnie populaires qui sont souvent en contact étroit avec des enfants avec lesquels l'hygiène n'est pas toujours strictement respectée.

Biliary Enterocytozoon bieneusi infection in a dog under immunomodulatory therapy.

An 8-year-old female spayed Labrador retriever was presented for the evaluation of severe weight loss 10 weeks after starting an immunomodulatory treatment, including prednisolone and cyclosporine, for meningoencephalitis of unknown origin. Plasma biochemistry analysis showed mild to moderate increases in liver enzyme activities and a moderate decrease in urea concentration. Abdominal ultrasound revealed mild hepatomegaly and a large gall bladder with unremarkable wall and content. Cholecystocentesis was performed and bile was examined both cytologically and by molecular methods, which revealed the presence of Enterocytozoon bieneusi. Treatment was initiated with albendazole but was discontinued due to the development of severe neutropenia. The medical management was subsequently changed to fenbendazole and the dog made a complete recovery. This report describes the first case of clinical manifestation and successful treatment of biliary E. bieneusi infection in a dog.

Neosporosis in Argentina: Past, present and future perspectives.

Neosporosis, caused by the protozoan Neospora caninum, was first diagnosed in Argentinean cattle in the 90's. With a national bovine stock of approximately 53 million head, the cattle industry is socially and economically relevant. Severe economic losses have been estimated at US$ 33 and 12 million annually in dairy and beef cattle, respectively. Approximately 9% of bovine abortions in the Buenos Aires province are caused by N. caninum. In 2001, the first isolation of N. caninum oocysts from feces of a naturally infected dog was performed in Argentina and named as NC-6 Argentina. Further strains were isolated from cattle (NC-Argentina LP1, NC-Argentina LP2) and axis deer (Axis axis, NC-Axis). Epidemiological studies revealed a high distribution of Neospora-infections not only in dairy but also in beef cattle, with seroprevalence rates of 16.6-88.8% and 0-73%, respectively. Several experimental infection studies in cattle have been carried out, as well as attempts to develop effective vaccines to avoid Neospora-abortions and transmission. However, no vaccine has proven successful for its use in daily practice. Reduction of seroprevalence, vertical transmission and Neospora-related abortions have been achieved in dairy farms by the use of selective breeding strategies and embryo transfer. Neospora-infections have been also detected in goats, sheep, deer, water buffaloes (Bubalus bubalis) and gray foxes (Lycalopex griseus). Moreover, Neospora-related reproductive losses were reported in small ruminants and deer species and could be more frequent than previously thought. Even though diagnostic methods have been improved during the last decades, control of neosporosis is still not optimal. The development of new strategies including new antiprotozoal drugs and vaccines is highly needed. This paper reviews the information from the previous 28 years of research of N. caninum in Argentina, including seroprevalence and epidemiological studies, available diagnostic techniques, experimental reproduction, immunization strategies, isolations and control measures in domestic and non-domestic animals from Argentina.

Strongyle faecal egg counts in Swiss horses: A retrospective analysis after the introduction of a selective treatment strategy.

The standard parasite management of horses based on regular anthelmintic treatments, now practiced for decades has resulted in a worrying expansion of resistant helminth populations, which may considerably impair control on the farm level. The aim of the present study was to obtain a retrospective (year 2010 - 2016) nationwide analysis of faecal egg count (FEC) data from the Swiss adult horse population, related to horse age and geographic region. Thirteen labs provided a total of 16,387 FEC data of horses aged four to 39 years (average: 13.6 years). The annual number of performed FEC tests increased from 38 to 4,939 within the observation period. Independent of the annual sample size the yearly patterns of the FEC were very similar. Seventy-eight percent (n = 12,840) of the samples were negative and 90 % (n = 14,720) showed a FEC below 200 strongyle eggs per gram (EPG) of faeces. The annual mean strongyle FEC ranged between 60 and 88 EPG with a total mean of 75 EPG. Horses aged 4-7 years showed a significantly (p < 0.00001) higher mean FEC compared with the other age groups, differences were not significant among the older horses. Based on ZIP codes, samples were allocated by 70.0 %, 6.0 % and 0.2 % to the German-, French- and Italian-speaking regions of Switzerland, respectively. With 222 EPG the mean FEC in the French part of Switzerland was significantly higher (p < 0.05) than in the German-speaking region (60 EPG). Eggs of Parascaris spp., anoplocephalids and Strongyloides westeri were found in 0.36 %, 0.32 % and 0.01 % of the samples, respectively. Based on 3,813 questionnaire feedbacks from owners in 2017 covering a total of 12,689 horses, sixty-eight percent (n = 8,476) were dewormed without diagnosis, two percent (n = 240) were not dewormed at all, whereas for 30 % (n = 3,721) the selective anthelmintic treatment (SAT) concept was applied. The SAT implementation rate differed significantly (p < 0.0005) between regions, with 33 %, 20 % and 25 % for the German-, French- and Italian-speaking areas, respectively. The rate of horses spending 16-24 h on pasture per day was significantly higher in the French-speaking region compared to the German-speaking part of Switzerland (p < 0.0001). In addition, pasture hygiene was practiced at a significantly lower rate in the French-speaking part compared to the German- and Italian-speaking regions (both p < 0.0001). Overall, the shift towards the SAT-concept represents a very promising development with respect to mitigating the further spread of anthelmintic resistance.

[Sacrocystis hjorti lesions in hunted red deer in Switzerland]. / Erster Nachweis von Sarcocystis hjorti bei der Fleischuntersuchung von Rothirschen in der Schweiz.

In meat samples from 2 hunted red deer (Cervus elaphus) of different origins (region Ilanz, region Filisur) large-scale greenish tissue discolorations with a gelatinous change of fascia were observed and diagnosed as eosinophilic fasciitis. Sarcocystis hjorti, a recently described Sarcocystis species in red deer and moose in Norway, was found as the causing agent. Foxes are regarded as final hosts in the development cycle of this parasite. Factors leading to such cases of eosinophilic fasciitis due to sarcosporidiosis, which is widespread in farm and wild ruminants and is normally inapparent are largely unknown. According to meat inspection directives carcasses with such discolorations have to be declared unfit for human consumption.

[First cases of besnoitiosis in cattle in Switzerland]. / Erste Fälle von Besnoitiose bei Rindern in der Schweiz.

Bovine besnoitiosis has been diagnosed in neighboring countries but not in Switzerland so far. This disease occurs endemically in France and focal outbreaks have been reported in Germany and Italy. To determine if Besnoitia besnoiti is introduced into Switzerland through the import of breeding cattle from France, a systematic serological survey was performed. A total of 412 breeding cattle (from 114 farms) imported from France into Switzerland between 2005 and 2011, were serologically examined for antibodies against B. besnoiti using a commercial ELISA kit (PrioCHECK© Besnoitia Ab 2.0, Prionics AG, Zurich, Switzerland). Sixty-four (15.5 %) animals reacted positive in ELISA. The serologic diagnosis was confirmed by an indirect immunfluorescence test (IFAT) and a Western blot (WB) in only 2 Limousin cows imported from France on a farm in Eastern Switzerland. Subsequently, this whole herd (n = 16) was examined clinically and serologically and 2 additional Limousin cows imported from Germany also reacted positive in the three serological tests. One of these cows presented B. besnoiti tissue cysts in the scleral conjunctiva and typical skin lesions in the head region. The infection was further confirmed cytologically, histopathologically and by PCR. It can be concluded that the parasite is most likely being introduced into Switzerland through the import of infected animals.

The golden jackal (Canis aureus): A new host for Echinococcus multilocularis and Trichinella britovi in Switzerland.

INTRODUCTION: The golden jackal (Canis aureus) is a wild canid new to Switzerland. It is an officially monitored species and all deceased individuals are submitted for post-mortem examination to collect baseline health data. This includes parasitological examinations, with an emphasis on zoonotic, reportable infections, such as those caused by Trichinella spp. or Echinococcus spp. From 2016 to 2021, five golden jackals originating from four Swiss cantons were submitted for full post-mortem examination. In one case only organ samples were available, and therefore parasitological examination was not possible. Parasite stages recovered during necropsy, as well as by routine coproscopical techniques, were morphologically identified. Taeniid eggs and adult tapeworms were processed for molecular species identification. Additionally, tongue and diaphragm were analysed for Trichinella spp. by the artificial digestion technique followed by multiplex-PCR in positive cases. Of the four jackals investigated for parasites, hookworm eggs were detected in one animal, both adult worms and eggs of Echinococcus multilocularis were present in another case, and one animal was free of parasites. Eggs of E. multilocularis as well as eggs of Toxocara canis and sporocysts of Sarcocystis sp. were detected in the intestinal content, and Trichinella britovi larvae were found in the muscle samples of the last case. The health monitoring programme in place for protected carnivores in Switzerland allowed us to add the golden jackal to the list of hosts for the endemic zoonotic parasites E. multilocularis and T. britovi in this country. Hunters, farmers, and other persons who could come in contact with golden jackals should be aware of the associated health risk and handle faeces and carcasses with caution.

INTRODUCTION: Le chacal doré (Canis aureus) est un canidé sauvage nouvellement présent en Suisse. Il s'agit d'une espèce officiellement surveillée et tous les individus morts sont soumis à un examen post-mortem afin de recueillir des données sanitaires de base. Cela inclut un examen parasitologique mettant l'accent sur les infections zoonotiques à déclaration obligatoire, telles que celles causées par Trichinella spp. ou Echinococcus spp. De 2016 à 2021, cinq chacals dorés originaires de quatre cantons suisses ont été soumis à un examen post-mortem complet. Dans un cas, seuls des échantillons d'organes ont été envoyés, l'examen parasitologique n'a pas été possible pour cet animal. Les stades parasitaires trouvés lors de l'examen pathologique et de la coprologie de routine ont été identifiés morphologiquement. Les espèces de ténias (œufs et stades adultes) ont été déterminées par des techniques de biologie moléculaire. En outre, la recherche de Trichinella spp. a été effectuée sur du tissu musculaire lingual et diaphragmatique par la technique de digestion artificielle suivie d'une PCR multiplex dans les cas positifs. Sur les quatre chacals ayant fait l'objet d'une recherche de parasites, des œufs d'ankylostomes ont été détectés chez un animal, des vers adultes et des œufs d'Echinococcus multilocularis étaient présents chez un autre animal, et aucun parasite n'a été trouvé dans un autre cas. Chez le dernier cas, des œufs d'E. multilocularis ainsi que des œufs de Toxocara canis et des sporocystes de Sarcocystis sp. ont été détectés dans le contenu intestinal, et des larves de Trichinella britovi ont été trouvées dans les échantillons de muscle. Le programme de surveillance sanitaire mis en place pour les carnivores protégés en Suisse a donc permis d'ajouter le chacal doré à la liste des hôtes des parasites zoonotiques endémiques E. multilocularis et T. britovi. Les chasseurs, agriculteurs et autres personnes susceptibles d'entrer en contact avec le chacal doré doivent être conscients du risque sanitaire associé et manipuler les fèces et les carcasses avec précaution.

First isolation of Neospora caninum from the faeces of a dog from Portugal.

We report the in vitro isolation of Neospora caninum from the faeces of a naturally infected 8-year-old male stray boxer from Portugal. Vero cell cultures were infected using parasite stages obtained after oral inoculation of gamma-interferon knockout mice with 10(2) sporulated oocysts. The isolate was identified by microscopical examination, as well as histological, immunological and molecular methods including a DNA-microsatellite-based typing technique, and was subsequently named NC-P1. The DNA-microsatellite pattern observed in the NC-P1 isolate was not previously reported for any N. caninum isolate. To our knowledge, this is the first isolation of N. caninum from the faeces of a naturally infected dog from Portugal.

Molecular comparison of Neospora caninum oocyst isolates from naturally infected dogs with cell culture-derived tachyzoites of the same isolates using nested polymerase chain reaction to amplify microsatellite markers.

Neospora caninum infection is an important cause of bovine abortion. The infection can be transmitted transplacentally or by ingestion of oocysts shed by definitive hosts. There are few reports of dogs naturally shedding N. caninum oocysts and only some oocyst isolates were transferred into cell culture. The aim of the present study was to analyse N. caninum oocysts from the faeces of naturally infected dogs using a microsatellite-based typing technique and to compare them with cell culture-derived tachyzoites of the same isolates. To this end, N. caninum oocysts from six naturally infected dogs were inoculated into gamma-interferon knockout mice. After these mice had developed disease, tissue samples or peritoneal washings from necropsied mice were transferred into cell culture. Nested-PCR techniques were developed for the sensitive and specific amplification of N. caninum microsatellite-containing regions (MS1B, MS2, MS3, MS4, MS5 and MS10). DNA was extracted from oocysts and cell culture tachyzoites of each isolate, followed by amplification and sequence analysis of microsatellite-containing regions. Each parasite isolate examined yielded a unique microsatellite genotype, while no differences were revealed when data for N. caninum oocysts were compared with cultured tachyzoites of the same isolate. Our technique may allow the typing of clinical samples and different strains of N. caninum at the molecular level. This method may prove useful for the identification of infection sources in molecular epidemiological studies.

Frequency of horizontal and vertical transmission for Sarcocystis cruzi and Neospora caninum in dairy cattle.

Sarcocystis cruzi and Neospora caninum infections in cattle are common throughout the world, and cause important economical losses. N. caninum can be transmitted horizontally by ingestion of oocysts or vertically from the infected dam to the fetus via the placenta. Vertical transmission for S. cruzi is infrequent and horizontal transmission is considered the most important route of infection. The objectives of this study were to evaluate the frequency of horizontal and vertical transmission for S. cruzi and N. caninum in a dairy cattle herd and to analyze IFAT titers as predictors of vertical transmission. Serum samples (n = 173) were collected from dairy calves at birth prior to colostrum ingestion and from their dams. In addition, 12 calves were also sampled after ingestion of colostrum, 25 female calves were sampled at 7 months, and 81 of the dams were also sampled at breeding. Sera were evaluated for S. cruzi and N. caninum antibodies by IFAT starting at 1:25 dilution. For S. cruzi, vertical transmission frequency was 1.7%, and all female calves evaluated at 7 months and cows were seropositive. Seroprevalence for N. caninum was 80.9% in cows and 30% in precolostrum calves. Vertical transmission frequency was 37.1%. Cows with high antibody titers (> or = 400) showed higher vertical transmission frequency (94.8%) than cows with low antibody titers (between 25 and 200) (14.8%). Negative precolostrum calves (7/12) had postcolostrum N. caninum titers 2-8 times higher than their dams. Estimated horizontal transmission frequency was 51 and 47%, based on differences of seroprevalences in calves and dams, and on the seroconversion of 9/19 negative precolostrum female calves when retested at 7 months, respectively. Average N. caninum titers of cows at breeding and calving were 120.6 and 320.9, respectively. Cows with a high titer at breeding had a high titer at calving. Therefore, N. caninum IFAT titers at breeding and calving could potentially be used as predictors of vertical transmission.

Isolation and molecular characterization of Toxoplasma gondii from captive slender-tailed meerkats (Suricata suricatta) with fatal toxoplasmosis in Argentina.

In this study, the diagnosis of fatal disseminated toxoplasmosis in three captive slender-tailed meerkats (Suricata suricatta) in the zoo of La Plata, Argentina and the invitro isolation and molecular characterization of Toxoplasma gondii are reported. The animals showed depression, dyspnea and hypothermia, and also ataxia in one case, and died within 1-5 days. The main histopathological lesions included interstitial pneumonia, non-suppurative inflammatory changes and focal necrosis in liver, spleen, kidney and brain. Tachyzoites or tissue cysts were present in lung, liver, spleen, brain, striated muscle, kidney, intestine and mesenteric lymph node sections, and stained strongly with T. gondii antiserum in immunohistochemical analysis. T. gondii was isolated in Swiss mice and in bovine monocytes cultures from tissues of one of the meerkats. The isolate was cryopreserved and it was named TG-Suricata-1. T. gondii DNA was demonstrated in tissues of all three animals and in tachyzoites isolated in cell cultures. The PCR-RFLP analysis of markers based in the loci 3'-SAG2, 5'-SAG2, BTUB, GRA6, SAG3, c22-8, L358, PK1, c29-2 and Apico of T. gondii produced patterns corresponding to the clonal type III. Type III strains of T. gondii possess no or only little virulence in the mouse model, however their association with virulence in other animal species is uncertain. In the present case, T. gondii of the clonal lineage III was responsible for fatal cases in S. suricatta. To our knowledge, this is the first report of isolation and genotyping of T. gondii from S. suricatta.

Toxoplasma gondii infection and toxoplasmosis in farm animals: Risk factors and economic impact.

The protozoan parasite Toxoplasma gondii is a zoonotic parasite that can be transmitted from animals to humans. Felids, including domestic cats, are definitive hosts that can shed oocysts with their feces. In addition to infections that occur by accidental oral uptake of food or water contaminated with oocysts, it is assumed that a large proportion of affected humans may have become infected by consuming meat or other animal products that contained infective parasitic stages of T. gondii. Since farm animals represent a direct source of infection for humans, but also a possible reservoir for the parasite, it is important to control T. gondii infections in livestock. Moreover, T. gondii may also be pathogenic to livestock where it could be responsible for considerable economic losses in some regions and particular farming systems, e.g. in areas where the small ruminant industry is relevant. This review aims to summarize actual knowledge on the prevalence and effects of infections with T. gondii in the most important livestock species and on the effects of toxoplasmosis on livestock. It also provides an overview on potential risk factors favoring infections of livestock with T. gondii. Knowledge on potential risk factors is prerequisite to implement effective biosecurity measures on farms to prevent T. gondii infections. Risk factors identified by many studies are cat-related, but also those associated with a potential contamination of fodder or water, and with access to a potentially contaminated environment. Published information on the costs T. gondii infections cause in livestock production, is scarce. The most recent peer reviewed reports from Great Britain and Uruguay suggest annual cost of about 5-15 million US $ per country. Since these estimates are outdated, future studies are needed to estimate the present costs due to toxoplasmosis in livestock. Further, the fact that T. gondii infections in livestock may affect human health needs to be considered and the respective costs should also be estimated, but this is beyond the scope of this article.

Seroprevalence of Neospora caninum, Toxoplasma gondii and Sarcocystis sp. in llamas (Lama glama) from Jujuy, Argentina.

Llamas (Lama glama) are South American camelids described as intermediate hosts of Neospora caninum, Toxoplasma gondii and Sarcocystis aucheniae. Due to the potential role of these protozoan infections as a cause of economic losses, the aim of this study was to determine the seroprevalence for T. gondii, N. caninum and Sarcocystis sp. in llamas from Argentina. Serum samples from 308 llamas (>2 years old) were collected between 2005 and 2007. A total of 55 farms located in six departments of Jujuy province, Argentina were sampled. Presence of antibodies to N. caninum, T. gondii and Sarcocystis sp. was determined by the indirect fluorescent antibody test (IFAT). For Sarcocystis, 2 different bradyzoites-based antigens were prepared using S. aucheniae and S. cruzi. Sera were tested at dilutions 1:25 and 1:50. Antibodies to N. caninum were found in 4.6% serum samples. Fifty percent of departments and 14.5% of farms had positive animals. Antibodies to T. gondii were found in 30% of samples, distributed in 66% of departments and 43.6% of farms. Antibodies to Sarcocystis sp. were detected in 96% of samples and all departments and farms had positive animals, suggesting frequent contact between llamas and canids. Co-infection with N. caninum, T. gondii and Sarcocystis sp. was also recorded. Low seroprevalence of N. caninum in llamas detected in this study could be related to climatic and geographical conditions that limit cattle breeding activity, reducing the source of infection for definitive hosts. Seroprevalence of T. gondii and the positive animal distribution suggest frequent contamination of grass with felid faeces. In conclusion, this is the first report of combined seroprevalence for N. caninum, T. gondii and Sarcocystis sp. in llamas. Further studies are needed to determine the potential role of these protozoan infections as cause of abortion in Argentina as well as presence of these protozoans in llama meat used for human consumption.

Analysis of Toxoplasma gondii clonal type-specific antibody reactions in experimentally infected turkeys and chickens.

Due to their ground-feeding behaviour, free-ranging chickens and turkeys are exposed to oocysts and are good indicators of the presence of Toxoplasma gondii in the environment. In addition, poultry may become infected by ingestion of tissues of infected intermediate hosts such as small rodents. Free-ranging poultry are considered an important source of T. gondii infection in humans, especially in developing countries. Knowledge on T. gondii genotypes in infected animals and humans is important for understanding the epidemiology of T. gondii infections. The aim of the present study was to analyse the ability of experimentally infected turkeys and chickens to develop a T. gondii clonal type-specific antibody response (IgY) after i.v. inoculation with tachyzoites of three T. gondii clonal lineages, types I, II and III. A peptide microarray displaying a panel of 101 different synthetic peptides was used for serotyping. Peptide sequences were derived from polymorphic regions of 16 T. gondii proteins (GRA1, GRA3-7, SAG1, SAG2A, SAG3, SAG4, SRS1, SRS2, ROP1, NTPase I and NTPase III and BSR4). The array was probed with 120 sera from experimentally infected chickens and turkeys inoculated with different doses of T. gondii tachyzoites (104, 103 and 102) collected from isolates representative for T. gondii clonal types I (RH), II (ME49) or III (NED) and uninfected controls. After screening of the peptides with reference sera from chickens and turkeys, and evaluation of data by Receiver Operating Characteristics analysis, 41 and 40 peptides were identified that appeared suitable to detect type-specific reactions with sera collected at 2, 5, 7 and 9 weeks p.i. Selected peptides allowed the identification of T. gondii clonal types, until 9 week p.i., which the chickens or turkeys had been inoculated with. At 9 weeks p.i., a high proportion of the experimentally infected chickens (67% (12/18)) and turkeys (61% (11/18)) no longer reacted with the selected peptides. Serotyping of the infection in individual chickens or turkeys was only possible when the whole peptide panel was applied. Clonal type-specific antibody responses were dynamic in both poultry species and depended on the individual animal and the time after infection.

Toxoplasmosis in captive Bennett's wallabies (Macropus rufogriseus) in Argentina.

Wallabies and other Australian marsupials are among the most susceptible species to Toxoplasma gondii. Fatal generalized toxoplasmosis was diagnosed in two captive 3 year-old female Bennett's wallabies (Macropus rufogriseus) from Argentina (w 1 and w 2) with a history of sudden death. Both animals had internal joeys which died 2 days after their mothers. Serologically, both females and one adult male without clinical signs from the same enclosure (w 3) had antibody titers for T. gondii>or=800 by the modified agglutination test (MAT); another adult male (w 4) was negative (MAT titer<25). Microscopically, tachyzoites were observed associated to non-suppurative meningoencephalitis, hepatitis, myositis, myocarditis and severe enteritis in hematoxylin and eosin stained sections from both w 1 and w 2. Immunohistochemically, parasites in heart, brain and liver sections of both female wallabies reacted with T. gondii antiserum. T. gondii was isolated from brain tissues of w 1 and w 2 by bioassay in mice and by culture in bovine monocytes and both isolates were cryopreserved. Genomic DNA was isolated from tachyzoites grown in cultures derived from both animals. The primer pair B22/B23 specific for T. gondii produced 115bp amplicons on poliacrylamide electrophoretic gels. Stray cats were suspected as the possible source of infection. Not all infected macropods were ill, showing that the infection may be asymptomatic and is not always fatal. A vertical infection could not be proved in the joey from w 2. As far as we know, this is the first confirmed report of toxoplasmosis in Bennet's wallabies in Argentina.