Molecular identification of Sarcocystis species in wild boar (Sus scrofa) and pigs (Sus scrofa domesticus) in Brazil.

Sarcocystis spp. are protozoan parasites that form cysts in the organs and musculature of various animal species. The species Sarcocystis miescheriana and Sarcocystis suihominis are pathogenic to pigs and wild boars (Sus scrofa), acting as intermediate hosts, while humans are the definitive host for S. suihominis. To date, there have been no reports of the identification of these coccidian species in Sus scrofa in Brazil. Therefore, in this study, we conducted the first molecular identification of Sarcocystis species using PCR-RFLP and sequencing. A total of 210 samples were analyzed, of this total, 67 tested positive for Sarcocystis spp., representing 31.9% of the total samples assessed. Out of the total positive samples, 55 (82.1%) were identified as S. miescheriana and 8 (11.9%) as S. suihominis, a zoonotic species. Additionally, other species related to bovines, such as S. cruzi and zoonotic S. hominis, were detected in 3.0% of the samples, serving as contaminants in the pork products. The presence of S. suihominis in swine and wild boar samples is concerning due to the zoonotic risk and potential environmental contamination, as humans act as definitive hosts, also for the presence of S. hominis as a bovine contaminant in pork sausages. Furthermore, we confirmed the efficacy of the PCR-RFLP technique as a reliable tool for the identification of Sarcocystis species, demonstrating its potential use in laboratories for molecular diagnosis and rapid identification of these parasites, aiming to protect public health and ensure food safety.

Sarcocystis in horses from Rio Grande do Sul, Brazil: Molecular identification of Sarcocystis bertrami and Sarcocystis neurona in muscle tissues.

Horses are intermediate hosts of Sarcocystis spp. capable of forming cysts in their musculature. This study aimed to detect sarcocysts and investigate the presence of nucleic acids from Sarcocystis spp. in samples of striated muscles from horses in the State of Rio Grande do Sul, Brazil, necropsied at the Veterinary Pathology Laboratory of the Federal University of Santa Maria. A total of 108 samples were collected from 24 horses and examined through direct examination. Microscopic tissue cysts were observed in three samples: tongue (2) and esophagus (1) from two animals. Extractions were performed on the found cysts and tissues, even though sarcocystosis detection was not present. DNA samples were subjected to Nested-PCR using Tg18s primers, and the amplified products were subjected to Restriction Fragment Length Polymorphism (RFLP) using DdeI and HpaII enzymes. DNA belonging to Sarcocystis spp. was amplified in tissues from 91.7% (22/24) of the equines, and 67.6% (73/108) of the samples tested positive in the Nested-PCR reaction. The tissues with the highest detection frequency were: diaphragm 92.3% (12/13), gluteal muscle 77.2% (17/22), and esophagus 66.7% (4/6). In RFLP, Sarcocystis spp. was detected in 21 tissues from 11/22 equines, and cysts, identified through nucleotide sequencing, were determined to be S. bertrami. S. neurona was identified in 11 samples from 7/22 animals, with co-infection detected in 5/22 cases. The high detection rate indicates a concerning circulation of the protozoan, particularly the zoonotic S. bertrami found in all tissues, which are commonly exported for human consumption.

Anti-Leishmania spp. antibody detection in domestic cats from a visceral leishmaniasis transmission area.

Feline leishmanial infection is reported worldwide, but the epidemiological role of domestic cats in the leishmaniasis cycle remains unclear, and cats might act as cryptic reservoir hosts in endemic areas with no feline leishmaniosis cases. Considering that, a serological screening for anti-Leishmania spp. antibodies was performed by indirect immunofluorescence antibody test (IFAT) in 389 necropsied cats' serum samples from a new visceral leishmaniasis transmission area with no feline leishmanial infection reported to unveil if the cats are being exposed to the parasite. The overall seroprevalence for Leishmania spp. was 11.05% (43/389). No association was found between sex, neutering status, age group, breed, coat length, feline immunodeficiency virus (FIV) infection, and Leishmania spp. antibody detection. A positive association was found with coat color (cats within the orange spectrum with white [particolor]) (OR = 2.47, CI 95% 1 - 6.13, P = 0.044) and a negative association (OR = 0.38, CI 95% 0.18 - 0.79, P = 0.01) between feline leukemia virus (FeLV) infection and IFAT positivity for Leishmania spp. Therefore, it is concluded that the seroprevalence found was greater than 10%, indicating contact of the protozoan with cats in the region served.

Sarcocyst Quantification and Viability: Freezing Treatment as an Alternative to Carcass Condemnation.

PURPOSE: The inspection of animal products is important for controlling parasitic zoonoses. Some processes that guarantee food safety to consumers such as carcass condemnation cause economic losses. This study aimed to detect Sarcocystis cysts in cattle hearts obtained from slaughterhouses and to evaluate sarcocyst viability after freezing treatment. METHODS: When myocardial tissues were minced and subjected to fresh examination, sarcocysts were observed in all analyzed tissues resulting in 21.73 cysts/g of tissue. Sarcocyst viability was verified after tissue freezing at 35 ± 2 °C and - 20 ± 2 °C for 0-12 h. After freezing, the tissues were minced, and sarcocysts were collected and stained with Tripan Blue. In addition, cysts were mechanically disrupted to check bradyzoite viability. RESULTS: Cysts and bradyzoites were unviable at - 35 °C for ≥ 3 h and - 20 °C for ≥ 8 h. CONCLUSION: These results suggest freezing treatment as an alternative to condemnation of cattle carcasses contaminated with Sarcocystis spp. Similar studies using freezing treatment with other animals infected by Sarcocystis must be conducted.

Abortion outbreak in a sheep flock caused by Toxoplasma gondii clonal type III.

Toxoplasma gondii is a major cause of reproductive losses in small ruminants in several countries. We describe here an outbreak of T. gondii-associated abortion in sheep in Southern Brazil. The flock was comprised of 55 adult sheep, and late-term abortions and stillbirths were detected in 15/36 (41.66%) gestating ewes. Serum samples collected from 45 sheep were tested for T. gondii through indirect immunofluorescence assay; IgM and IgG positive results were detected in 44.44% (20/45) and 86.67% (39/45) of the cases, respectively. Four fetuses and two placentas were pathologically evaluated. Gross changes were restricted to fetal membranes and were characterized by multifocal white areas in the cotyledons. Microscopically, these areas corresponded to necrotic foci affecting the chorionic epithelium accompanied by rare cysts of T. gondii. The main histological change in fetal tissues consisted of well-demarked and sparsely distributed necrotic foci in the central nervous system. Tissue samples from all four fetuses and one placenta had positive PCR results for T. gondii. Restriction fragment length polymorphism (RFLP) genotyping using ten markers (SAG1, 5'-3'SAG2, alt.SAG2, SGA3, BTUB, GRA6, c22-8, c29-2, L358, and PK1) was carried out on one sample, and results were consistent with T. gondii clonal type III (ToxoDB-PCR-RFLP genotype #2, TgCpBr4).

Detection of Sarcocystis spp. and Toxoplasma gondii in swine and detection of DNA of these protozoa in tissues and sausages / Soroprevalência de Sarcocystis spp. e Toxoplasma gondii em suínos e detecção do DNA desses protozoários em tecidos e embutidos

Abstract The seroprevalence of Sarcocystis spp. and Toxoplasma gondii was researched in swine raised in Santa Maria, RS, Brazil. Serum samples from 84 pigs from 31 farms were tested using indirect immunofluorescence assay (IFA) for both agents. Additionally, 53 samples of pork sausages and tissues destined for human consumption, including: salami, sausage, black pudding, heart, tongue, brain, and rib muscle, were submitted to PCR to detect DNA for each agent. The frequency of anti-Sarcocystis spp. antibodies was 36.9% (31/84), with titers ranging from 32 to 1024, and 25% (21/84) for anti-T. gondii antibodies, with titers ranging from 64 to 2048. Sarcocystis spp. and T. gondii DNA were detected in 67.9% (36/53) and 13.2% (7/53) of samples, respectively. The presence of antibodies and the detection of DNA from Sarcocystis spp., and T. gondii suggests that the pigs were infected and may serve as an important reservoir for both parasites. The infection by these protozoa in the swine population is relevant to public health due to their zoonotic potential.

Resumo A soroprevalência de Sarcocystis spp. e Toxoplasma gondii foi pesquisada em suínos criados em Santa Maria, RS, Brasil. Amostras de soro de 84 suínos de 31 fazendas foram testadas pela reação deimunofluorescência indireta (IFA) para ambos os agentes. Adicionalmente, 53 amostras de embutidos suínos e tecidos cárneos destinados ao consumo humano, incluindo: salame, linguiça, morcela, coração, língua, cérebro e músculo da costela foram submetidas à PCR para detecção de DNA para cada agente. A frequência de anticorpos anti-Sarcocystis spp. foi de 36,9% (31/84), com títulos variando de 32 a 1.024; e 25% (21/84) para anticorpos anti-T. gondii, com títulos variando de 64 a 2048. A presença de DNA de Sarcocystis spp. e T. gondii foi detectada em 67,9% (36/53) e 13,2% (7/53) das amostras avaliadas, respectivamente. A detecção de anticorpos e DNA de Sarcocystis spp. e T. gondii sugere que os suínos foram infectados e podem servir como um importante reservatório de ambos os parasitas. A circulação desses agentes na população suína é relevante para a saúde pública devido ao seu potencial zoonótico.

Immunogenicity in sheep of Uruguayan commercial vaccines against bovine alphaherpesvirus 1, 5 and bovine pestiviruses / Imunogenicidade em ovelhas de vacinas comerciais Uruguaias para alfaherpesvírus bovino 1, 5 e pestivírus de bovinos

ABSTRACT: The serological responses induced by four commercial inactivated Uruguayan vaccines against bovine alphaherpesviruses (BoHV)-1 and -5 and bovine pestiviruses (BVDV-1, BVDV-2, and HoBiPeV) were evaluated in sheep. Thirty-seven sheep were immunized twice (day 0 and 25) and their serum samples were tested at different intervals (days 0, 25, 40, 60, and 90) post-vaccination (PV). Among the four vaccines tested, only one (G4) could induce the production of moderate neutralizing antibody titers against BoHV-1 and -5 and BVDV-1 and -2. The G3 vaccine showed a neutralizing serological response against the bovine alphaherpesviruses only. The G1 and G2 vaccines produced extremely low levels of antibodies in a few vaccinated animals only (geometric mean titers (GMT) 2.2). Similar levels of immunological responses were induced by the G4 vaccine against BoHV-1 and -5, and titers of neutralizing antibodies induced in approximately 70% of the animals are known to confer protection (GMT > 8). For bovine pestiviruses, the vaccine stimulated response of G4 against BVDV-2 was higher compared to that against BVDV-1, and extremely low for HoBiPeV. The peak of neutralizing antibodies to BoHV-1 and BVDV-1 was observed on days 40 and 60 PV, respectively. Thereafter, a remarkably decrease in neutralizing antibody response was observed at day 90 PV. These results demonstrated that tested commercial Uruguayan vaccines did not induce a serological response of adequate magnitude and duration. Thus, it is important to periodically review formulations and compositions of commercial vaccines against bovine alphaherpesviruses and pestiviruses.

RESUMO: A resposta sorológica induzida por quatro vacinas comerciais uruguaias inativadas contra os alfaherpesvírus bovinos (BoHV-1 e -5) e pestivírus de bovinos (BVDV-1, BVDV-2 e HoBiPeV) foi avaliada em ovinos. Os animais foram imunizados duas vezes (dia 0 e dia 25) e o soro testado em diferentes intervalos (dias 0, 25, 40, 60 e 90) após a vacinação (PV). Dentre as quatro vacinas testadas, apenas uma (G4) apresentou títulos de anticorpos neutralizantes moderados para os BoHV-1 e -5, BVDV-1 e 2. A vacina G3 apresentou resposta somente para alfaherpesvírus bovinos. As vacinas G1 e G2 estimularam resposta somente em alguns animais vacinados. Para a vacina G4, observou-se que a resposta imunológica frente ao BoHV-1 e 5 foi semelhante e pelo menos 70% dos animais apresentaram níveis protetivos de anticorpos neutralizantes. Para os pestivírus bovinos, a vacina G4 estimulou resposta para o BVDV-2 mais elevada quando comparada com o BVDV-1, e quase que indetectável para HoBiPeV. O pico de anticorpos neutralizantes para o BoHV-1 foi observado no dia 40 PV e no dia 60 PV para o BVDV-1. Após isso, observou-se um decréscimo considerável na resposta de anticorpos neutralizantes. Os resultados demonstraram que vacinas comerciais uruguaias testadas não induziram resposta sorológica de magnitude e duração adequadas. Assim, ressalva-se a importância de rever periodicamente a formulação e composição das vacinas comerciais para alfaherpesvírus e pestivírus bovinos.