Genetic characterization of Toxoplasma gondii from human and chicken isolates from Argentina.

This study aimed to determine the nPCR-RFLP genotypes of newly obtained T. gondii isolates from human congenital toxoplasmosis cases in Argentina and to determine their allelic profiles for virulence genes ROP18/ROP5. In addition, the ROP18/ROP5 profiles were also determined for previously characterized T. gondii samples. Isolation from congenital toxoplasmosis cases was carried out in mouse bioassay from two placentas (P1 and P2). Genotyping for the new human isolates was performed by nPCR-RFLP using 10 markers. The samples analyzed for ROP18/ROP5 included the two newly obtained isolates (from the congenital toxoplasmosis cases) and nine previously genotyped T. gondii DNA samples from humans and chickens. The results for P1 and P2 named as TgHm18-02Arg and TgHm19-01Arg showed ToxoDB genotypes #14 (non-archetypal) and #2 (clonal type III), respectively. Non-archetypal #14 has been isolated from human cases before in Argentina. However, this is the first report of T. gondii clonal type III in a human case in the country. The ROP18/ROP5 combination was detected in nine samples: 3/3 (n = 1), 4/3 (n = 4), 4/4 (n = 3), and 3-4/4 (n = 1). Notably, the 4/4 profile was identified for the first time and exclusively in T. gondii samples from Misiones province (which borders southern Brazil). Further studies are required to corroborate the regionalization of the ROP18/ROP5 profiles in Argentina.

Identification of Sarcocystis spp. in wild boars (Sus scrofa) from Argentina.

Sarcocystis spp. are intracellular protozoan parasites with an obligatory heteroxenous life cycle. The objective of this study is to identify Sarcocystis spp. in wild boar muscles from Argentina by light and transmission electron microscopy and molecular characterization. Muscle samples from diaphragm, tongue, masseter, intercostals, heart, and forelimbs of 240 wild boars were analyzed. Of the animals, 48.3% (116/240) were positive for sarcocysts by light microscopy, whereas 45.8% (110/240) were positive for Sarcocystis spp. by PCR targeting 18S rRNA fragment. These samples were subjected to a specific PCR for S. suihominis coxI gene, 3.6% (4/110) of which were weak positives. Unfortunately, sequence analysis was inconclusive. This could be related to a potentially low S. suihominis cyst load in the samples, or to an incomplete primer matching with the South American S. suihominis sequences. Seventeen individual sarcocysts were positive by PCR for the 18S rRNA fragment, whose sequences showed 99.75-100% identity with each other and with previously reported S. miescheriana sequences. A total of 21 cysts collected from 11 muscle samples and analyzed by TEM presented a cyst wall type compatible with S. miescheriana, and one cyst presented an ultrastructure compatible with S. suihominis. The latter came from a sample that also contained S. miescheriana cysts, indicating that the animal was co-infected. This is the first study that provides infection rates and describes and identifies morphological and molecular features of Sarcocystis spp. cysts in wild boars from South America.

Identification of Sarcocystis spp. in synanthropic (Muridae) and wild (Cricetidae) rodents from Argentina.

The occurrence of Sarcocystis species was investigated in synanthropic (Muridae) and wild (Cricetidae) rodents from Argentina. Nine species were captured (n = 356). Sarcocysts were detected in muscles of 8.7% (31/356) and 3.7% (4/106) of the rodents by histopathology and direct microscopic observation, respectively. PCR-sequencing targeting the 18S rRNA, cox1, and ITS1 regions was performed on samples with positive histopathology. Four different 18S rRNA sequences or sequence groups with high intra-group identities (99.6-100%) were detected in Mus musculus, Oxymycterus rufus, Akodon azarae, and Necromys lasiurus. Eight sequences showed 99.5-99.7% identity with S. dispersa. Thirteen sequences showed low identity (95.3-96.4%) with other Sarcocystis spp. The obtained coxI sequences (n = 9) were almost identical to each other and showed a high similarity with S. strixi (99.2-99.5%) and S. lutrae (99.1%), despite the 18S rRNA sequences from the same samples suggested the occurrence of at least two species. This suggests that coxI may not show high variability in Sarcocystis spp. that use rodents as intermediate hosts. Six ITS1 sequences were obtained, showing high identity but low coverage with several Sarcocystis spp. Multilocus sequence typing and BLAST analysis did not lead to an accurate species identification. Possible reasons are the detection of new species or the limited molecular information available from previously described Sarcocystis spp. Phylogeny suggests that the detected Sarcocystis spp. may use raptor birds or snakes as definitive hosts. This study represents the first molecular identification of Sarcocystis spp. in naturally infected rodents of the Cricetidae and Muridae families in South America.

Fatal sarcocystosis in psittacine birds from Argentina.

Five psittacine birds, one eastern rosella (Platycercus eximius), one rose-ringed parakeet (Psittacula krameri), two eclectus parrot (Eclectus roratus), and one princess parrot (Polytelis alexandrae), all housed in a commercial aviary from La Plata, Buenos Aires, Argentina, suddenly died after a short period of dyspnea. The most significant histopathological findings for all specimens were interstitial exudative pneumonia, with marked congestion and hemorrhage, septa thickening, and massive perivascular lymphoplasmacytic infiltration. Structures compatible with protozoal schizonts were observed in the capillary lumen. No bacterial development was obtained and the real-time PCR for Chlamydia spp. and several psittacine viruses were negative. All the samples resulted negative on the specific PCR for T. gondii. Sarcocystis spp. PCR was positive in the lung and/or liver samples from all birds. The samples showed a restriction pattern of S. neurona and of S. falcatula-like by PCR-RFLP using JNB25-JD396 and JNB33-JNB54 primers, respectively. Sequences obtained from Sarcocystis sp. 18S rRNA and COI gene from 4 birds showed a high identity among them. The 18S rRNA fragment and complete gene sequences obtained showed the highest similarity with S. falcatula and S. speeri sequences but also with S. neurona SN5 isolate sequence. Likewise, COI sequences have 99.89-100% similarity with S. falcatula and S. speeri sequences. Based on all biological and molecular information recorded, we conclude that the etiological agent was S. falcatula-like, close related with the species shed by opossums in South America.

Epidemic abortions due to Neospora caninum infection in farmed red deer (Cervus elaphus).

This study describes for the first time an abortion outbreak caused by Neospora caninum in farmed red deer. During a 5-year period, farmed hinds, naturally mated, were regularly ultrasound monitored to detect reproductive losses over their gestation. During the 4 years previous to the outbreak, abortion rates ranged from 4.7 to 8.6% (average 6.5%), and serology for indirect diagnosis of neosporosis and toxoplasmosis was performed. At the fifth year, the abortion rate increased to 25.3%. During this outbreak, three aborted foetuses and their placentas were recovered and submitted to laboratory for etiological diagnosis. Blood samples were collected from the 81 hinds at the end of the gestational period and the seropositivity rate for N. caninum, Toxoplasma gondii, Brucella abortus, bovine viral diarrhoea virus and bovine alphaherpesvirus type 1 was 66.7%, 67.9%, 0.0%, 8.6% and 0.0%, respectively. Neospora caninum-seropositive hinds (OR = 5.7, P = 0.0271) and hinds with high antibody titres to N. caninum (OR = 7.4, P = 0.0130) were more likely to abort than seronegative hinds. In addition, N. caninum seropositivity rate in the aborted hinds was higher (OR = 5.4, P = 0.033) than the non-aborted hinds. No association was found between T. gondii nor BVDV-seropositivity and abortions. Typical protozoal histopathologic findings (necrotizing non suppurative encephalitis, meningitis, myocarditis, hepatitis, among others) were observed in all foetuses. Neospora caninum was immunolabelled by immunohistochemistry in several tissues from two foetuses, and infection was also confirmed in the three foetuses by serology and/or DNA detection. No other abortifacient agent was detected in the foetuses. Their dams showed high N. caninum antibody titres (≥ 6400). Serologic evidence and epidemiological data recorded suggested a point-source of N. caninum infection before the occurrence of the outbreak, probably related with contaminated feedstuff with oocysts. Moreover, the intensive production system with a high stocking rate could be also considered a factor which might have increased the risk of horizontal N. caninum infection in this herd.

Neospora caninum and Toxoplasma gondii infections and their relationship with reproductive losses in farmed red deer (Cervus elaphus).

The aims of the present study were to determine the Neospora caninum and Toxoplasma gondii seropositivity rates in farmed red deer hinds from Argentina and their relationship with reproductive losses. Over a 2-year period, 449 hinds from 4 commercial farms were serologically tested at late gestation for N. caninum and T. gondii by IFAT. During the first year, a sequential serological analysis was carried out at 3 different time points to analyze antibody dynamics from mating until the end of the gestation period. Fetal and postnatal mortality rates were estimated by 3 successive ultrasound scannings (us) annually and a breeding control carried out after the calving period. Ultrasound fetal measurements were used to estimate conception date and gestational age of abortions. The seropositivity rate for N. caninum was 25.5% (37/145) for the yearlings and 34.2% (104/304) for the adults, while for T. gondii was 64.3% (93/145) and 78.3% (238/304), respectively. Abortions detected at us1 and us2 were 13/21 (61.9%) with a range of gestational age of 30-87 days, while abortions detected at us3 were 8/21 (38.1%) with a range of gestational age of 49-209 days. The fetal mortality rate was 4% and 5.8%, while the postnatal mortality rate was 18.8% and 4.1% of 101 yearlings and 294 adult pregnant hinds, respectively. Most seropositive hinds to both protozoans showed a stable antibody titer pattern from mating to the end of gestation, and a lower proportion developed an increase in titers suggesting infection recrudescence. Seroconversion during the gestational period was demonstrated in 6 and 50 hinds for N. caninum and T. gondii, respectively. Hinds with fetal mortality were more likely to be seropositive to N. caninum (OR = 3.1) or have N. caninum titers ≥400 (OR = 27.4) than hinds that weaned a fawn. No statistical associations were detected for T. gondii seropositivity and reproductive losses. The pregnancy rate was not affected by N. caninum or T. gondii infection, while the serological evidence of N. caninum causing postnatal mortality was marginal. Based on serological evidence, N. caninum would be a potential abortigenic agent in red deer hinds.

Evaluation of biological behavior of Toxoplasma gondii atypical isolates # 14 and # 163.

Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting warm-blooded animals, including humans. A highly diverse genetic population has been reported in Central and South America, predominating mainly atypical genotypes. Different genotypes showed different biological behavior in mice. The aim of this study was to evaluate the biological behavior of T. gondii isolates obtained from Macropus rufogriseus (TgMr) and Saimiri boliviensis (TgSb) identified as atypical genotypes # 14 and # 163, respectively. Strains RH, ME49 and VEG were used as reference for clonal types I, II and III, respectively. In vitro invasion and replication capacity assays were analyzed at 6 and 18 hpi, respectively. In vivo assay was performed in Swiss mice (n = 30) using 1 × 102 and 1 × 103 parasites/mouse as infective doses (ME49, VEG, TgMr, TgSb and negative control). Morbi-mortality and tissues PCR were assessed. Lymphoproliferation assays were performed and gamma interferon was measured by ELISA. The ME49 strain showed the highest invasion, followed by TgSb and VEG, while RH and TgMr presented the lowest invasions. The RH strain and the TgSb isolate showed more endodyogeny events (fastest doubling times) than VEG and ME49 strains and the TgMr isolate. Both atypical isolates showed high virulence (100% morbi-mortality, at 8-10 dpi) and parasite DNA was detected in all tissue samples. Splenocytes from mice inoculated with TgMr and TgSb registered the highest values of gamma interferon. An in vitro invasion-replication index was established which correlates inversely with virulence in mice. In conclusion, T. gondii atypical isolates # 14 and # 163 showed a different in vitro behavior than clonal strains, with low invasion-replication indexes but being highly virulent in mouse model.

Sarcocystis spp. infection in South American deer huemul (Hippocamelus bisulcus) and pudu (Pudu puda) from Patagonian National Parks, Argentina.

Sarcocystis spp. are intracellular protozoan parasites with heteroxenous life cycles. This study described Sarcocystis spp. infection in adult South American native deer huemul (Hippocamelus bisulcus) and pudu (Pudu puda). Heart, diaphragm, tongue, and skeletal muscle samples were collected from 5 huemuls and 2 pudus, found dead in National Parks. Direct microscopic examination, transmission electron microscopy, PCR, and sequencing were performed. Sarcocystis spp. microscopic thin-walled cysts were identified in 3 huemuls and 1 pudu. Several cysts from 1 huemul and 1 pudu were observed by TEM; ultrastructure was similar to previously reported as cyst wall type 17 and types 2 and 8, respectively. Fragments of the 18S rRNA and cytochrome c oxidase subunit I (cox1) genes were amplified and sequenced from 3 individual cysts from 2 huemuls and 2 cysts from the pudu. The sequences from huemuls showed a high identity among them (> 99%) at both amplified targets. The highest identities were > 99.7% at 18S rRNA and 93% at cox1 with S. tarandivulpes sequences. The 18S rRNA gene sequences from pudus showed an identity > 99.7% with Sarcocystis sp., S. taeniata, and S. linearis sequences, while the cox1 sequences were different, one showing 99.42% identity with S. venatoria and the other 98.22% with S. linearis. A single species, similar to S. tarandivulpes, was identified in all huemul samples while 2 molecularly different Sarcocystis spp. were found in 1 pudu with high similarities to either S. venatoria or to S. linearis, S. taeniata-like, and S. morae. Based on the cox1 sequence identities, at least the Sarcocystis sp. in huemuls might represent a new species, primarily occurring in this host. Additional sarcocyst isolates from both hosts need to be examined molecularly in order to firmly establish whether these species are indeed native to huemuls and/or pudus or are derived from introduced deer species.

An Ibero-American inter-laboratory trial to evaluate serological tests for the detection of anti-Neospora caninum antibodies in cattle.

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero-American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1-7) and three enzyme-linked immunosorbent assays (ELISAs 1-3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the "Pre-test information," which uses previous epidemiological and serological data; (2) the "Majority of tests," which classifies a serum as positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2.

Sarcocystis rommeli, n. sp. (Apicomplexa: Sarcocystidae) from Cattle (Bos taurus) and its Differentiation from Sarcocystis hominis.

Cattle (Bos taurus) are intermediate hosts for three named species of Sarcocystis, S. cruzi, S. hirsuta, and S. hominis. Recently, a fourth species was identified and named S. sinensis. However, S. sinensis originally named a species of Sarcocystis in water buffalo (Bubalus bubalis) in China. Based on unverifiable evidence, it was suggested that the same parasite infects cattle. In addition, S. sinensis was recently declared as nomen nudum because its naming violated the rules of International Code of Zoological Nomenclature. Thus, the fourth species using cattle as an intermediate host does not have a valid name. Here, we propose a new name, Sarcocystis rommeli for the S. sinensis-like parasite from cattle in Argentina, and differentiate it ultrastructurally from S. hominis sarcocysts from experimentally infected cattle. Sarcocystis rommeli sarcocysts were microscopic with a 5-µm-thick wall with slender villar protrusions (Vp); the Vp were up to 5 µm long, up to 0.5 µm wide, and of uneven thickness, often bent at an angle. The ground substance layer (Gs) was up to 0.8 µm thick and smooth. Vesicular structures were seen at the base of the Vp. The bradyzoites were 10-12 µm long. Sarcocystis hominis sarcocysts had Vp that were often upright, up to 7.5 µm long, and up to 1.8 µm wide; the Gs was up to 2 µm thick and without vesicles. Its sarcocyst wall was up to 5.6 µm thick, the vp were bent at an angle, up to 5.8 µm long, the Gs was up to 2 µm thick, but without vesicles seen in S. rommeli. Beef containing sarcocysts of S. rommeli was not orally infectious for two human volunteers and a red fox (Vulpes vulpes). The Sarcocystis described here is molecularly different from S. cruzi, S. hirsuta, and S. hominis based on 18S rRNA and cox1 gene sequences.

Sarcocystis masoni, n. sp. (Apicomplexa: Sarcocystidae), and redescription of Sarcocystis aucheniae from llama (Lama glama), guanaco (Lama guanicoe) and alpaca (Vicugna pacos).

There is considerable confusion concerning the species of Sarcocystis in South American camelids (SAC). Several species names have been used; however, proper descriptions are lacking. In the present paper, we redescribe the macroscopic sarcocyst forming Sarcocystis aucheniae and describe and propose a new name, Sarcocystis masoni for the microscopic sarcocyst forming species. Muscles samples were obtained from llamas (Lama glama) and guanacos (Lama guanicoe) from Argentina and from alpacas (Vicugna pacos) and llamas from Peru. Individual sarcocysts were processed by optical and electron microscopy, and molecular studies. Microscopic sarcocysts of S. masoni were up to 800 µm long and 35-95 µm wide, the sarcocyst wall was 2·5-3·5 µm thick, and had conical to cylindrical villar protrusions (vp) with several microtubules. Each vp had 11 or more rows of knob-like projections. Seven 18S rRNA gene sequences obtained from sarcocysts revealed 95-96% identity with other Sarcocystis spp. sequences reported in the GenBank. Sarcocysts of S. aucheniae were macroscopic, up to 1·2 cm long and surrounded by a dense and laminar 50 µm thick secondary cyst wall. The sarcocyst wall was up to 10 µm thick, and had branched vp, appearing like cauliflower. Comparison of the 11 sequences obtained from individual macroscopic cysts evidenced a 98-99% of sequence homology with other S. aucheniae sequences. In conclusion, 2 morphologically and molecularly different Sarcocystis species, S. masoni (microscopic cysts) and S. aucheniae (macroscopic cysts), were identified affecting different SAC from Argentina and Peru.

Toxoplasma gondii isolates from chickens in an area with human toxoplasmic retinochoroiditis.

The aim of this study was to detect, isolate and genetically characterize Toxoplasma gondii from tissues obtained from free range chickens which were breed in farms from patients with toxoplasmic retinochoroiditis in Misiones, Argentina. Thirty three samples of head (refrigerated = 18 and frozen = 15) from free range chickens were processed. Refrigerated (n = 18) chicken central nervous systems (CNS) were bioassay in mice. DNA was obtained from all samples (n = 33) and PCR was performed using TOX5-TOX8 T. gondii specific primers. Positive PCR samples were characterized by nested-PCR and restriction fragment length polymorphism using the markers SAG2, BTUB, GRA6, SAG3, PK1, L358, C22-8, C29-2 and Apico. T. gondii DNA was amplified in 30.3% (10/33) of CNS samples. Isolates were obtained in 27.7% (5/18) of inoculated CNS samples (TgCk11-9Arg, TgCk13-5Arg, TgCk14-5Arg, TgCk14-6Arg and TgCk14-7Arg). Seven samples showed a restriction pattern to all markers and were identified as atypical with several alleles type III. Genotyping of T. gondii from samples of patients with retinochoroiditis in the same area could improve the understanding of the epidemiology of toxoplasmosis in the region.

Sarcocystosis in wild red deer (Cervus elaphus) in Patagonia, Argentina.

Sarcocystis spp. are protozoan parasites with a heteroxenous life cycle, which produce cysts in the muscle of herbivorous animals. In these animal species, sarcocystosis is frequently asymptomatic, although it may occur with high prevalence. Seven Sarcocystis spp. have been described in red deer (Cervus elephus). The aim of this study was to determine the prevalence of sarcocystosis, and to perform the morphological and molecular characterization of Sarcocystis spp. found in wild red deer of the Nahuel Huapi National Park (NHNP), Patagonia, Argentina. Full necropsies of 62 red deer killed by hunters in the NHNP and neighboring areas were performed. Samples of heart and skeletal muscle were examined histologically and selected samples were also examined by transmission electron microscopy (TEM), PCR and sequencing. Sarcocystis spp. thin walled cysts were detected in 62 % (38/62) of heart, and in 22 % (3/14) of skeletal muscle samples examined histologically. TEM revealed a smooth and thin cyst wall (≤1 µm), with scarce and separated ribbon-like protrusions. A total of three partial and one full 18S ribosomal RNA (rRNA) gene sequences were obtained, and showed the highest identity (≥99 %) with Sarcocystis taeniata, a species described in moose (Alces alces). The morphological and molecular results indicate that red deer in Argentina are frequently infected with S. taeniata, a species for which the definitive host is unknown. The present results also confirm that Sarcocystis spp. using cervids as intermediate host are not host-specific. Further studies are needed to improve the epidemiological knowledge of Sarcocystosis in red deer.

Evaluation and comparison of serological methods for the detection of bovine neosporosis in Argentina.

Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. The accurate diagnosis of neosporosis is essential for management and control measures. The aims of this study were: i) to evaluate the performance of an in-house enzyme-linked immunosorbent assay based on the 38kDa native antigen (p38-ELISA) to diagnose bovine neosporosis in Argentina using a well- characterized local sera panel from experimentally infected and naturally exposed cattle and ii) to compare the diagnostic performance and agreement of three N. caninum serological tests: p38-ELISA, indirect fluorescence antibody test (IFAT) and immunoblotting (IB) using the same sera panel. Serum samples testing either positive or negative by IFAT and IB were considered "Relative Standards of Comparison" (RSC) and used for p38-ELISA evaluation. Receiver operating characteristics analysis revealed that p38-ELISA was highly accurate (area under the curve= 0.982) according to RSC with a cut-off index of 0.0905. Relative sensitivity and specificity of p38-ELISA were 97.8% and 99.5%, respectively and agreement between RSC and p38-ELISA was almost perfect (k= 0.97). The evaluation and performance comparison of serological tests were performed according to the definition of gold standard based on the decision of the "majority of tests". All tests displayed high sensitivity and specificity values (greater than 95%); and excellent agreement. This study describes the accurate performance of p38-ELISA evaluated locally and the highly accurate diagnostic performance of the studied tests for the detection of anti-N. caninum antibodies in cattle from Argentina.

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 was investigated. 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 snakes and rodents as definitive and intermediate hosts (IH), respectively. Sequence data and also the results of clinical and parasitological examinations suggest that the snakes were 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.

Early Detection and Management of Lamanema chavezi infection in a llama (Lama glama) in Switzerland.

Lamanema chavezi is an entero-hepatic strongylid parasite specific to South American camelids. It has been reported only on few occasions outside South America. Due to its hepatic migration, it can cause extensive liver damage, leading to granulomatous and fibrotic hepatitis and manifesting with lethargy, anorexia, and even death. We are reporting the second case of L. chavezi infection in Europe and the first in Switzerland. The patient was a three-year old neutered male llama (Lama glama). Clinical examination revealed bloody mucous discharge from the anus. Fecal sedimentation/flotation revealed strongylid eggs consistent with L. chavezi, which were molecularly confirmed by a PCR targeting the ITS2 plus 5.8S and 28S rDNA flanking regions and amplicon sequencing. Eighteen weeks after administration of a single dose of eprinomectin (0.2 mg/kg i.m.), no further L. chavezi eggs were detected in the feces. The source of infection could not be traced back. The entire herd consisted of llamas bred in Switzerland. L. chavezi has been rarely reported outside South America, but its potential for pathogenicity and establishment should not be underestimated. Fecal sedimentation/flotation techniques should be routinely performed to ensure early detection of the parasite.

Morphological and molecular characterization of Sarcocystis spp. in pigs (Sus scrofa domestica) from Argentina.

Sarcocystis spp. are intracellular protozoan parasites with an obligatory heteroxenous life cycle. The objective of this study was to identify Sarcocystis spp. in pig muscles from Argentina, by light and transmission electron microscopy (TEM), and molecular studies. Muscles samples from 561 pigs (Sus scrofa domestica) were classified according to the breeding system in: intensive farming (IF, n = 295; animals kept in confinement during most of their productive cycle), or semi-extensive farming (SEF, n = 266; animals bred outdoors, generally family or backyard production). Results showed that 24.8% (139/561) were positive by light microscopy, with a significantly higher prevalence in the SEF (34.6%; 92/266) than the IF pigs (15.9%; 47/295) (p < 0.05). Of the 202 samples analyzed by PCR, 96 were positive (47.5%) for the 18S rRNA (18S ribosomal RNA) fragment. All samples analyzed by the S. suihominis specific coxI (mitochondrial cytochrome c oxidase subunit I) PCR (n = 235; 96 positives by 18S rRNA PCR and 139 positives by light microscopy) were negative. Fourteen individual cysts were positive for the 18S rRNA PCR and sequenced. Consensus sequences obtained from the 18S rRNA fragment PCR ranged from 613 to 880 bp and showed 100% of identity between them and with previously reported S. miescheriana sequences. In all the pig samples analyzed by TEM, cyst wall ultrastructure was compatible with S. miescheriana. This is the first study that provides infection rates and describes and identifies morphological and molecular features of Sarcocystis spp. cysts in pigs from Argentina.

Angiostrongylus dujardini infection in a coconut lorikeet (Trichoglossus haematodus) from a zoological garden in Switzerland.

Angiostrongylus spp. (Metastrongyloidea) can cause severe disease in several animal species and humans. This report describes an infection with Angiostrongylus dujardini in a captive coconut lorikeet (Trichoglossus haematodus) from a zoo in Switzerland. The bird was reported being attacked by conspecifics, removed from the flock, and hospitalized. It showed lethargy, moderately reduced body condition, and lack of reaction to visual stimuli. Analgesic and antibiotic treatment were initiated but because of worsening of its general condition, the bird was euthanized the following day. Necropsy revealed multifocal, subcutaneous hemorrhages, diffusely reddened lungs and a moderately dilated right heart with several intraluminal nematodes embedded in a coagulum. Four worms were collected and microscopically examined. They were identified as adult females, measuring 19-21 mm long x 0.4-0.5 mm wide, with general morphological and morphometric characteristics consistent with angiostrongylid nematodes. In lung sections, multifocal collection of thin-walled embryonated eggs in variable stages of development was observed along with fully developed nematode larvae within the lumina of alveoli and lung vessels. Associated granulomatous infiltrates indicated a severe, multifocal, chronic, granulomatous pneumonia. The diagnosis of A. dujardini infection was formulated by morphological examination of adult and larval stages, supported by molecular analysis (PCR-amplification and sequencing of the ITS2, 5.8S and 28S rDNA flanking regions). This is the first report of A. dujardini infection in an avian species, providing evidence that birds can serve as accidental hosts of this parasite in addition to mammals, and that the parasite can reach maturity and multiply in the avian cardiorespiratory system.

Nested PCR effective to detect low viral loads of SARS-CoV-2 in animal samples.

SARS-CoV-2 emerged from an animal source and was then transmitted to humans, causing the COVID-19 pandemic. Since a wide range of animals are susceptible to SARS-CoV-2 infection, the zoonotic potential of SARS-CoV-2 increases with every new animal infected. The molecular gold standard assay for SARS-CoV-2 detection is real-time RT-PCR, where the Ct obtained is proportional to the amount of nucleic acid and can be a semi-quantitative measure of the viral load. However, since the use of real-time RT-PCR assays in animal samples is low due to the high costs, the use of validated nested PCR assays will help to monitor large-scale animal samplings, by reducing the costs of detection. In the present study, 140 samples from dogs and cats (15 SARS-CoV-2-positive samples with Ct values from 27 to 33, and 125 negative samples), previously analyzed by real-time RT-PCR, were analyzed by nested PCR. To increase the number of positive samples to determine the sensitivity of the assay, 40 human samples obtained during COVID-19 diagnosis in 2020 were included. The specificity of the primers was analyzed against samples positive to canine coronavirus (CCV) and feline infectious peritonitis virus (FIPV). To calculate the limit of detection (LoD) of the nested PCR, the viral load was estimated extrapolating the Ct value obtained by real-time RT-PCR. The Ct values obtained were considered as semi-quantitative and were able to distinguish between high, moderate and low viral loads. The Kappa value or "agreement" between assays and reliability of the nested PCR were also determined. Eleven of the animal samples analyzed by nested PCR targeting the N gene were detected as positive, while 129 were detected as negative to the virus, with Ct values ranging between17 and 31.5. All the samples from humans analyzed by nested PCR were positive. These results indicate that the assay has a sensitivity of near 95 % and a specificity of 100 %. No unspecific reactions analyzed by nested PCR were observed with the samples positive to CCV and FIPV. The samples detected as positive to SARS-CoV-2 by nested PCR were those that presented a Ct between17 and 31.5. The LoD of the nested PCR was estimated close to 50 copies/µL of viral load, corresponding with a Ct of 31.5. The Kappa value between assays was excellent (k = 0.829). The results obtained demonstrate that nested PCR is useful to detect SARS-CoV-2 low viral loads at a lower cost than with real-time RT-PCR.

Zoonotic nematode in the city of La Plata, Argentina: Report of a case of Calodium hepaticum in Rattus rattus.

This study describes a case of Calodium hepaticum (Trichinellida: Capillariidae) infection in an adult rat (Rattus rattus) from the periurban area of the city of La Plata in the province of Buenos Aires, Argentina. The rat was found with neurological signs (ataxia, lethargy, and episodes of unresponsiveness) in the food storage of a goat production facility. The liver was observed with hepatomegaly and diffuse and irregular yellowish-white spots appearing in striae or small nodules on the external surface and inside the liver. Subsequent microscopic and histopathological studies were performed. Eggs were observed by direct microscopy of the impression smear of liver tissue. A multifocal granulomatous tissue reaction with different stages of fibrocellular tissue was observed in the liver parenchyma. The granulomas contained adults and degenerated eggs delimited by an intense infiltrate of mononuclear cells. Macro and microscopic observations and histopathological liver lesions were compatible with C. hepaticum infection. To our knowledge, this is the first confirmation of C. hepaticum infection in R. rattus in Argentina, increasing the host record of this parasite and a new record of distribution in goat production systems in the country.