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.

Detection of Sarcocystis hominis, Sarcocystis bovifelis, Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis sigmoideus sp. nov. in carcasses affected by bovine eosinophilic myositis.

Bovine eosinophilic myositis is an inflammatory myopathy characterized by multiple focal or diffuse grey to green patches leading to condemnation of affected carcasses. Although its etiology is still uncertain, there is evidence that Sarcocystis species may play a role in the development of eosinophilic myositis. The goal of the present study was to identify Sarcocystis spp. in intralesional and extralesional tissues of condemned cattle carcasses, in order to evaluate the possible role of different bovine Sarcocystis spp. in the etiology of bovine eosinophilic myositis. Muscle samples (n = 100) of 26 affected carcasses were collected in Northern Italy. One to five samples with lesions and two aliquots of tissue without lesions were collected from each carcass; lesions were grossly categorized in green focal lesions and green diffuse patches. Genomic DNA was extracted and analyzed by multiplex-PCR targeting different Sarcocystis spp. Unidentified species were characterized morphologically (light microscopy, histology), ultrastructurally (scanning and transmission electron microscopy) and on the molecular level (complete 18S rRNA gene and partial cox1 gene sequencing). A bovine eosinophilic myositis prevalence of 0.017% was visually assessed by routine carcass inspection between 2014 and 2019 in Italy (184/1,108,150 slaughtered cattle). Out of 26 carcasses, 25 revealed the presence of at least one Sarcocystis species (96.2%). The presence of Sarcocystis spp. DNA was significantly more frequent in intralesional than in extralesional samples. Considering the different species, Sarcocystis bovifelis and Sarcocystis hominis were significantly more frequent in intralesional (41.7% and 50%, respectively) than in extralesional samples (1.9% and 15.4%, respectively), while there was no significant difference between the presence of Sarcocystis cruzi and Sarcocystis hirsuta in intralesional (27.1% and 2.1%, respectively) and extralesional (30.8% and 1.9%, respectively) samples. The presence of an unnamed Sarcocystis sp. showing thick-walled (3.7-5.4 µm) cysts with densely packed, flattened, undulating and narrow protrusions, which showed an S-shape in side view, was recorded in the diaphragm of two carcasses. Genomic DNA from individual sarcocysts isolated from the diaphragm was successfully amplified and further sequenced. Sequence comparison revealed <94.6% and 83.4% identity at 18S rRNA and cox1 genes, respectively, with other named Sarcocystis spp., while the phylogenetic analysis clearly separated the unnamed Sarcocystis sp. from the other Sarcocystis spp. using cattle as intermediate hosts. The present study contributes to the understanding of the importance of different Sarcocystis spp. in the pathogenesis of bovine eosinophilic myositis. The results emphasize the association of Sarcocystis hominis and Sarcocystis bovifelis with bovine eosinophilic myositis and highlight the presence of a new Sarcocystis sp. using cattle as intermediate hosts. The name Sarcocystis sigmoideus sp. nov. is proposed for the newly described Sarcocystis species.

Seroprevalence of Toxoplasma gondii in wild boars (Sus scrofa) hunted in Ukraine.

Toxoplasma gondii is an important zoonotic parasite worldwide, but it has received limited attention in Ukraine. A seroepidemiological study was conducted and samples from 452 wild boars that had been hunted in 2006-2011 in 23 of the 25 regions of Ukraine were tested to estimate T. gondii seroprevalence. A locally available commercial enzyme-linked immunosorbent assay (ELISA) was used for the investigation. Additionally, we tested 92 of the sera using a widely used commercial multi-species ELISA and an indirect immunofluorescence antibody test (IFAT). With the locally available ELISA, 35 of the 452 wild boars tested positive, yielding a seroprevalence estimate of 7.7% (95% confidence interval 5.5-10.5). The seropositive wild boars originated from eight of the regions. Using the majority criteria, 10/92 samples tested using both ELISAs and the IFAT were considered positive, yielding an estimated seroprevalence of 10.9% within the subset of samples. The highest seroprevalence was observed in wild boars hunted in Luhans'k (30.0%), Odesa (17.7%) and Kharkiv (12.7%). Seroprevalence was higher in older animals (13.3% for age group >12 months and 7.7% for age group ≤12 months). This is the first seroepidemiological study of T. gondii in wild boars in Ukraine. Assuming that seropositivity indicates presence of infectious parasites in the tissues, eating undercooked meat of wild boars hunted in Ukraine could be a potential source of infection to other hosts, including humans.

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.

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.

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.

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.

What is the role of Swiss domestic cats in environmental contamination with Echinococcus multilocularis eggs?

BACKGROUND: The role of the domestic cat as definitive host for Echinococcus multilocularis and thus in environmental contamination with eggs has not yet been entirely resolved. This study aimed to assess the prevalence of E. multilocularis and other gastrointestinal parasites in Swiss domestic cats and to compare the diagnostic sensitivity of different methods for the detection of intestinal taeniid infection. METHODS: Faecal samples from 146 cats were included in the study. Faecal samples only were available from 55 cats; for the other 91 cats, necropsy was performed in addition to faecal sample testing. All (n = 146) faecal samples were analysed by a combined sedimentation/flotation technique (44% ZnCl2) and by the sodium acetate-acetic acid-formalin (SAF) sedimentation technique; when sufficient material was available (n = 121 samples) the Baermann-Wetzel technique was also used. Additionally, all samples were analysed by two coproantigen (copro)-quantitative PCRs (qPCR): (i) a multiplex qPCR able to detect and differentiate between E. multilocularis, Echinococcus granulosus sensu lato and Taenia spp./other cestodes (CEST-qPCR) and (ii) an E. multilocularis-specific qPCR (EM-qPCR). Finally, the intestines were examined macroscopically and microscopically for parasite stages at necropsy (n = 91) and using an intestinal scraping technique (IST) (n = 64). RESULTS: Of the 146 cats examined, 24 (17.1%) were infected by intestinal parasites, namely Hydatigera (syn. Taenia) taeniaeformis (8.9%), Toxocara cati (6.1%), Capillaria sp. (3.4%), hookworms (3.4%), Mesocestoides litteratus (1.4%), Giardia sp. (1.4%), Cystoisospora rivolta (1.4%), Cystoisospora felis (0.7%), Toxoplasma gondii (0.7%), Hammondia hammondi (0.7%) and Strongyloides sp. (0.7%). Necropsy and the IST revealed adult H. taeniaeformis in 12 animals, of which eight faecal samples were positive by the CEST-qPCR (sensitivity = 67%) and six samples by the sedimentation/flotation technique (sensitivity = 50%). No E. multilocularis infection was detected in the sampled cats. Using Bayesian latent class analysis, the mean posterior prevalence probability was 0.0% (95% confidence interval 0-0.83%) for E. multilocularis. CONCLUSIONS: There was no evidence of E. multilocularis infection among the 146 cats examined, suggesting that the prevalence of this parasite is low (< 1%) in the Swiss domestic cat population. Nonetheless, some of the sampled cats were infected by parasites that have rodents as intermediate hosts, demonstrating successful predation by these cats, and some were infected with zoonotic parasites. Cats therefore should not be disregarded as potential hosts for E. multilocularis and other zoonotic parasites.

First report of Sarcocystis spp. (Apicomplexa, Sarcocystidae) in Lagostomus maximus (Desmarest, 1917) (Rodentia, Chinchillidae) in Argentina.

Sarcocystis is a genus of intracellular parasitic protozoa that infects various species of mammals, birds, and reptiles worldwide. At least 46 Sarcocystis species naturally infect rodents as intermediate hosts producing tissue cysts. This study aimed to provide the first report and molecular characterisation of Sarcocystis spp. in muscles from plains viscacha (Lagostomus maximus) in Argentina. Muscle samples of 53 plains viscachas from three provinces of Argentina were processed by homogenisation and optical microscopy to detect tissue cysts. Positive samples were analysed by PCR-sequencing, using the following markers: 18S rRNA, ITS1, and coxI. The 18S rRNA and coxI consensus sequences were aligned with other sequences from Sarcocystis spp., and phylogenetic trees were constructed. Of all animals processed, 13.2% (7/53) harboured Sarcocystis sp. cysts. 18S rRNA consensus sequences were obtained from four muscle samples and one individual cyst, and they showed 99.88-100% similarity, except for the cyst sequence, which showed 97.11% homology. Similarities of only 96-97% were recorded in the 18S rRNA fragment with other Sarcocystis spp. whose sequences are available in the GenBank. The five coxI fragment sequences obtained were 100% identical and showed an identity of 99.41-99.48% with S. canis. For ITS1 only short and low-quality sequences were obtained. In the phylogenetic trees, all the sequences from plains viscachas were positioned together in a branch separated from other Sarcocystis spp. These results could be related to new Sarcocystis spp. producing sarcocysts in plains viscachas. Besides, comprehensive cyst morphological analysis using TEM from the new Sarcocystis species will allow a description of the cyst wall ultrastructure. In this sense, further studies are needed to deepen these findings and elucidate other potential intermediate and possible definitive hosts.

High prevalence rates of Toxoplasma gondii in cat-hunted small mammals - Evidence for parasite induced behavioural manipulation in the natural environment?

Toxoplasma gondii causes one of the most frequent parasitic infections in vertebrates on earth. The present study aimed to assess the occurrence of T. gondii infection in cat-hunted wild small mammals, and to determine the circulating T. gondii genotypes in cat prey. There is evidence suggesting that T. gondii may manipulate rodents' behaviour enhancing transmission to their definitive feline host by facilitating predation. Given that most studies focusing on rodent behavior have been performed under laboratory conditions, we tested this hypothesis in the natural environment. We analysed 157 cat-hunted wild small mammals of six different species from Switzerland. Brain and skeletal muscle samples from each animal were tested for T. gondii DNA by PCR, and positive samples were genotyped using a multilocus sequence typing approach, including 10 genetic markers. Additionally, to evaluate exposure to cat faeces, the presence of Taenia taeniaeformis metacestodes was investigated at necropsy. The prevalence of T. gondii in cat-hunted Arvicola amphibius s.l. was 11.1% (7/63), 14.6% (7/48) in Apodemus spp., 13.6% (3/22) in Myodes glareolus, 6.7% (1/15) in Crocidura russula, and 0% in Microtus arvalis (0/8) and Sorex sp. (0/1). All completely genotyped T. gondii parasites, exhibited the ToxoDB #3 genotype, a Type II variant. We additionally analysed 48 trap-captured A. amphibius s.l., which all tested negative for T. gondii infection, contrasting with the higher prevalence in cat-hunted A. amphibius s.l. (0% vs. 11.1%; p = 0.0176). Furthermore, T. taeniaeformis was detected in both groups, indicating widespread contamination with cat faeces in the sampled areas. These results provide evidence that T. gondii infected rodents are at higher risk to be predated by cats and therewith support the behaviour manipulation hypothesis.

Identification of a new Sarcocystis sp. in marsh deer (Blastocerus dichotomus) from wetlands of Argentina.

The marsh deer (Blastocerus dichotomus) is the largest South American native deer species and is listed as "Vulnerable" by IUCN due to the population reduction. As part of a conservation and disease surveillance program, muscle samples from 14 marsh deer found dead in 2016 and 2017 in northeast Argentina were obtained at necropsy. Samples from each animal were processed as pooled muscles (heart, diaphragm, tongue and hindlimb) by homogenization and direct microscopical observation to detect intracellular Sarcocystis spp. cysts. Sarcocysts were observed in six samples, and several cysts recovered from two samples were processed by transmission electron microscopy. The cysts were thin-walled and showed a cyst-wall ultrastructure with ribbon-like protrusions similar to other species using cervids as intermediate host and canids as definitive hosts. Genomic DNA from individual sarcocysts from three marsh deer were successfully amplified by PCR of 18S rRNA and COI gene fragments and further sequenced. Sequence comparison revealed a 99.3-100% identity among them and only 93.7-96.6% and 88.8-89.7% identity at 18S rRNA and COI markers, respectively, with other Sarcocystis spp. Despite morphological similarities, the high sequence divergence at 18S rRNA and COI fragments allowed the assumption that Sarcocystis sp. from marsh deer is a different species from others using cervids as intermediate hosts. Therefore, we propose the name Sarcocystis blastoceris n. sp. for the species infecting marsh deer.

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.

Serological evidence of SARS-CoV-2 infection in pets naturally exposed during the COVID-19 outbreak in Argentina.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has rapidly spread worldwide. The monitoring of animals has shown that certain species may be susceptible to be infected with the virus. The present study aimed to evaluate the presence of SARS-CoV-2 antibodies by ELISA and virus neutralization (VN) in pets from owners previously confirmed as COVID-19-positive in Argentina. Serum samples of 38 pets (seven cats and 31 dogs) were obtained for SARS-CoV-2 antibody detection. Three out of the seven cats and 14 out of the 31 dogs were positive for SARS-CoV-2 by ELISA, and one cat and six dogs showed the presence of neutralizing antibodies in which the cat and two of the six dogs showed high titers. Another dog from which three serum samples had been obtained within eight months from the diagnosis of its owner showed the presence of antibodies at different times by both ELISA and VN. However, the results showed that the antibodies decreased slightly from the first to the third sample. Our results provide evidence that SARS-CoV-2 infection in pets living with COVID-19-positive humans from Argentina during the outbreak of SARS-CoV-2 can be detected by serology assay.

Association of bovine viral diarrhea virus, bovine herpesvirus 1, and Neospora caninum with late embryonic losses in highly supplemented grazing dairy cows.

The objectives of this study were: 1- to evaluate the association of Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BoHV-1), and Neospora caninum (N. caninum) with the risk for Late Embryonic Loss (LEL) in grazing dairy cows, 2- to evaluate blood progesterone concentration at the time of LEL occurrence, and 3- to describe a novel ultrasound-guided technique for conceptus sampling. We run a prospective cohort study involving 92 cows (46 LEL and 46 NLEL). An LEL cow was that having an embryo with no heartbeat, detached membranes, or floating structures, including embryo remnants detected at pregnancy check by ultrasonography (US) 28-42 days post-AI, whereas an NLEL cow was that with embryo heartbeats detectable by US at pregnancy check 28-42 d post-IA. We took two blood samples from every cow at pregnancy check by US (the day of LEL detection) and 28 d later to perform serological diagnosis of BVDV, BoHV-1, and N. caninum; and to measure blood progesterone concentration at pregnancy check (28-42 d post-AI). We also sampled the conceptus from all the LEL cows. We performed PCR to detect BVDV, BoHV-1, and N. caninum in sampled conceptuses from LEL cows. Finally, we evaluated the associations of risk factors (serological titers, seroconversion, and progesterone) with LEL odds with logistic models. The risk for LEL was associated with serological titers to BVDV (P = 0.03) and tended to be associated with seroconversion to BVDV, given that 19.6% (9/46) in LEL and 6.5% (3/46) in NLEL cows seroconverted to BVDV (P = 0.09). In addition, BVDV was detected in conceptuses from LEL cows that seroconverted to BVDV but not in LEL cows that did not seroconvert. Conversely, the risk for LEL was not associated with the titers or seroconversion to BoHV-1 and N. caninum. BoHV-1 and N. caninum were not identified in any of the conceptuses. Finally, blood progesterone concentration was similar in LEL and NLEL cows, and it was not associated with the risk for LEL (P = 0.54). In conclusion, BVDV infection is a risk factor for LEL in dairy cows.

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.

Pampas fox (Lycalopex gymnocercus) of the Argentine Pampas as intermediate host for Neospora caninum.

The Pampas fox (Lycalopex gymnocercus) is the most abundant wild canid from South America. This wild canid inhabits grasslands, open woodlands, and areas highly modified by extensive ranching and agricultural activities. We aimed to evaluate Neospora caninum infection in tissues from the Pampas fox from Argentina. A total of 41 free-living Pampas foxes were sampled in rural areas located in the Humid Pampas region, Argentina. Brain tissue and different muscles were assessed by histologic and molecular methods. No N. caninum cysts were observed in brain and muscle tissue samples analyzed by histology and immunohistochemistry. Molecular N. caninum identification from brain tissue was based on amplification by PCR of Nc-5 gene and ITS1 rRNA fragments and subsequent sequencing. The presence of N. caninum DNA was 74% (23/31) for the Nc-5 gene and was confirmed by a second ITS1 PCR in 55% (17/31) of the brain tested. Thirteen ITS1 consensus sequences were obtained, and all have a 99.58-100% similarity with N. caninum reference sequences. Only 4% (1/23) of muscles samples analyzed were positive for the Nc-5 gene of N. caninum. This study demonstrated a high prevalence of N. caninum DNA in brain from free-ranging Pampas fox of the Pampa Argentine, thus confirming that this wild canid is a wide distributed intermediate host.

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.

Sarcocystis neurona and related Sarcocystis spp. shed by opossums (Didelphis spp.) in South America.

Protozoan parasites of the genus Sarcocystis are obligatory heteroxenous cyst-forming coccidia that infect a wide variety of animals and encompass approximately 200 described species. At least four Sarcocystis spp. (S. falcatula, S. neurona, S. lindsayi and S. speeri) use opossums (Didelphis spp.) as definitive hosts, and two of them, S. neurona and S. falcatula, are known to cause disease in horses and birds, respectively. Opossums are restricted to the Americas, but their distribution in the Americas is heterogeneous. Five Didelphis spp. are distributed in South America (D. aurita, D. albiventris, D. marsupialis, D. imperfecta and D. pernigra) whereas just one opossum species (D. virginiana) is found in North America. Studies conducted in the last decades show that Sarcocystis spp., derived from South American Didelphis spp., have biological and genetic differences in relation to Sarcocystis spp. shed by the North American opossum D. virginiana. The aim of this review was to address the peculiar scenario of Sarcocystis species shed by South American opossums, with a special focus on diagnosis, epidemiology, and animal infections, as well as the genetic characteristics of these parasites.

First detection and molecular analysis of SARS-CoV-2 from a naturally infected cat from Argentina.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has rapidly spread worldwide. Studies of transmission of the virus carried out in animals have suggested that certain animals may be susceptible to infection with SARS-CoV-2. The aim of the present study was to investigate the infection of SARS-CoV-2 in pets (18 cats and 20 dogs) from owners previously confirmed as COVID-19-positive. Oropharyngeal and rectal swabs were taken and analyzed by real-time RT-PCR assays, while blood samples were taken for antibody detection. Of the total pets analyzed, one cat was found reactive to SARS-CoV-2 by real-time RT-PCR of an oropharyngeal and a rectal swab. This cat presented only sneezing as a clinical sign. Serological analysis confirmed the presence of antibodies in the serum sample from this cat, as well as in the serum from another cat non-reactive to real-time RT-PCR. Complete sequence and phylogenetic analysis allowed determining that the SARS-CoV-2 genome belonged to the B.1.499 lineage. This lineage has been reported in different provinces of Argentina, mainly in the Metropolitan Area of Buenos Aires. This study notifies the first detection of the natural infection and molecular analysis of SARS-CoV-2 in a cat from Argentina whose owner where COVID-19-positive. Although there is currently no evidence that cats can spread COVID-19, results suggest that health authorities should test pets with COVID-19-positive owners.

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.