Detection of ectoparasites and investigation of infection by Rickettsia spp. and Trypanosoma spp. in bats from Rio Grande do Sul, Brazil.

This study aimed to investigate the presence of ectoparasites and the occurrence of natural infection by Rickettsia spp. and Trypanosoma spp. in bats from Rio Grande do Sul (RS), Brazil. The evaluated animals were obtained from the Instituto de Pesquisas Veterinárias Desidério Finamor, sent by the Centro Estadual de Vigilância Sanitária, to carry out rabies diagnostic tests, during the period from 2016 to 2021. The bats came from 34 municipalities in RS. Of the 109 animals surveyed, 35.8% (39/109) had 385 ectoparasites, with an average of 9.9 parasites per animal. Of these bats, all had insectivorous feeding habits, with 35.9% (14/39) females and 64.1% (25/39) males. The co-parasitism of Chirnyssoides sp., Ewingana inaequalis, and Chiroptonyssus robustipes on Molossus currentium (Mammalia, Chiroptera) was recorded for the first time. All bats surveyed were negative for infection by the protozoan and bacteria. Thus, the expansion of the occurrence of these ectoparasites in insectivorous bats in RS was observed. Furthermore, this study corresponds to the first recorded interspecific associations for the species.

Sheep abortions associated with Neospora caninum and Toxoplasma gondii infections in multiple flocks from Southern Brazil.

Neosporosis and toxoplasmosis are important parasitic causes of abortions in small ruminants. This study verified the occurrence of these diseases in sheep fetuses from Santa Catarina State, Southern Brazil from 2015 to 2022. Sheep fetuses were necropsied with organ sampling for histopathology, and polymerase chain reaction (PCR) for Neospora caninum and Toxoplasma gondii using the Nc5 and SAG2 targets, respectively, in frozen brain tissue. Microbiological culture and RT-PCR for Pestivirus were conducted to discard other abortion causes. One positive fetus for toxoplasmosis was genotyped using multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) with ten genetic markers. Fifty-five sheep fetuses were evaluated, with 10 (18.2%) cases of neosporosis and 7 (12.7%) cases of toxoplasmosis, comprising six and four flocks, respectively. Macroscopically, neosporosis abortions exhibited fetal mummification, maceration, and arthrogryposis. Toxoplasmosis abortions showed fetal mummification and maceration. The neosporosis abortions included lymphoplasmacytic myositis (70%; 7/10) and myocarditis (60%; 6/10), in addition to necrotizing encephalitis and gliosis (50%; 5/10). Toxoplasmosis abortions included lymphoplasmacytic necrotizing encephalitis (71.4%; 5/7), lymphoplasmacytic myositis (42.8%, 3/7), and myocarditis (14.3%; 1/7). Through PCR, N. caninum and T. gondii were detected in 6 (60%) and 5 (71.4%) fetuses, respectively. In one fetus, T. gondii genotyping was conducted, which was characterized as atypical genotype ToxoDB #98. All of the cases were negative for Pestivirus and bacterial agents. This study establishes the occurrence of these diseases as causes of abortions, malformations, mummification, and fetal maceration in sheep, with the characterization of an atypical T. gondii genotype in one of the fetuses.

Detection of anti-Leishmania spp. antibodies in poultry from central region of Rio Grande do Sul, Brazil.

Domestic birds such as Gallus gallus, Meleagris gallopavo, Anser anser and Numida meleagris are widely distributed throughout the world and maintain contact with humans and other animal species considered reservoirs of both Visceral Leishmaniasis (VL) and American Tegumentary Leishmaniasis (ATL), including dogs and cats; wild canids, marsupials; and synanthropic animals such as rodents and chiroptera. Therefore, this study aimed to detect the presence of anti-Leishmania spp. antibodies in birds from a rural area of the municipality of Santa Maria, southern Brazil. From May to December 2022, 262 blood samples were collected from 244 chickens, 8 turkeys, 7 guinea fowl and 3 geese, distributed in 27 rural properties in 6 districts. All the sites visited presented positive birds for the presence of Leishmania spp. Thus, it is inferred that, contact with this protozoan can induce the production of antibodies, suggesting that these animals can be used as sentinels for the circulation of this agent. In addition, the blood of these animals is a preferred food source for insects of the subfamily Phlebotominae, which can be used them as bioindicators of the presence of these phlebotomes.

Increased frequency of detection of anti-Toxoplasma gondii antibodies in domestic cats after outbreak of human toxoplasmosis.

Toxoplasmosis affects various organisms, including humans. In 2018, the largest outbreak of human toxoplasmosis described so far was reported in southern Brazil, with 809 human cases reported, and water as the potentially primary source of infection. Therefore, in this study, we aimed to evaluate the seroprevalence of Toxoplasma gondii in naturally infected domestic cats before and after the human toxoplasmosis outbreak, as well as the potential for environmental contamination by the number of cats infected after the outbreak. We evaluated 381 serum samples from domestic cats in southern Brazil, using an indirect immunofluorescence assay, with samples considered positive at a titer of 1:20. We found that 73% (204/279) and 27% (75/279) of the samples analyzed before the outbreak were negative and positive, respectively. After the outbreak, 62% (69/112) were negative of the samples were and 38% (43/112) were positive. Notably, the proportion of positive samples before the outbreak before (27%) was significantly lower than that after the outbreak (38%; P = 0.020). Therefore, the increased seroprevalence of T. gondii in cats was probably correlated with the ingestion of contaminated water. Therefore, it is important to monitor animals, mainly definitive hosts, after toxoplasmosis outbreaks, considering that these animals can contaminate the environment and, consequently, humans.

Five years of the biggest outbreak of human toxoplasmosis in Santa Maria, Brazil: a review.

Toxoplasmosis is a parasitic disease caused by infection with the protozoan Toxoplasma gondii. In 2018, the first cases of people with clinical signs of acute febrile syndrome were reported, and in the same year, the largest outbreak of human toxoplasmosis ever described in the literature was reported. In this sense, the present work sought to describe the evolution of the outbreak cases in the municipality of Santa Maria, Rio Grande do Sul State, Brazil, as well as the studies conducted and published during and after the outbreak in the municipality (the period between 2018 and 2023). In addition, the discussion of public policies and their modifications after the notification of this outbreak. As a result of this research, verifying the evolution of notified and confirmed cases, the possibility of detection and genotypic characterization of T. gondii and the possibility of co-infections and evaluation of the humoral response is possible. With regard to public policies, the importance of detecting the agent through the heel prick test and increasing the monitoring of water quality to prevent outbreaks.

Detection of anti-Trypanosoma spp. antibodies in cattle from southern Brazil.

Bovine trypanosomosis, caused by Trypanosoma vivax, is a disease that originated in Africa and currently affects cattle in several South American countries, including almost all Brazilian states. Despite the reports on T. vivax infection in southern Brazil, data on its circulation status is currently unavailable. In this study, we aimed to detect anti-Trypanosoma spp. IgG antibodies in cattle from Rio Grande do Sul and suggest areas with T. vivax transmission risk. A total of 691 serum samples from cattle in the intermediate regions of Rio Grande do Sul were analyzed using indirect immunofluorescence assay (IFA). The overall seroprevalence of anti-Trypanosoma antibodies in cattle was 24.6% (170/691). The detection rate ranged from 0-37.3%, with a high prevalence in the intermediate regions of Ijuí (37.3%), Uruguaiana (30.7%), and Passo Fundo (28.9%). Thus, these regions were suggested as possible bovine trypanosomosis risk areas due to the high seroprevalence. This is the first serological study to determine Trypanosoma spp. infection status in cattle from Rio Grande do Sul, providing data on the epidemiology of trypanosomosis in the state.

Identification of infection by Leishmania spp. in wild and domestic animals in Brazil: a systematic review with meta-analysis (2001-2021).

Leishmaniasis is a zoonosis caused by protozoan species of the genus Leishmania. It generates different clinical manifestations in humans and animals, and it infects multiple hosts. Leishmania parasites are transmitted by sandfly vectors. The main objective of this systematic review was to identify the host, or reservoir animal species, of Leishmania spp., with the exception of domestic dogs, that were recorded in Brazil. This review included identification of diagnostic methods, and the species of protozoan circulating in the country. For this purpose, a literature search was conducted across index journals. This study covered the period from 2001 to 2021, and 124 studies were selected. Eleven orders possible hosts were identified, including 229 mammalian species. Perissodactyla had the highest number of infected individuals (30.69%, 925/3014), with the highest occurrence in horses. In Brazil, the most commonly infected species were found to be: horses, domestic cats, rodents, and marsupials. Bats, that were infected by one or more protozoan species, were identified as potential reservoirs of Leishmania spp. Molecular tests were the most commonly used diagnostic methods (94 studies). Many studies have detected Leishmania spp. (n = 1422): Leishmania (Leishmania) infantum (n = 705), Leishmania (Viannia) braziliensis (n = 319), and Leishmania (Leishmania) amazonensis (n = 141). Recognizing the species of animals involved in the epidemiology and biological cycle of the protozoan is important, as this allows for the identification of environmental biomarkers, knowledge of Leishmania species can improve the control zoonotic leishmaniasis.

Production performance and economic analysis of broiler chickens after vaccination with a live attenuated vaccine against avian coccidiosis.

The objective of this study was to evaluate the effectiveness of live attenuated commercial vaccine LIVACOX® T against avian coccidiosis upon parasite exposure through its correlation with productive and economic performance, clinical observation, and oocyst excretion of broiler chickens. For this purpose, 420 1-day-old Cobb chicks were divided into five groups of 84 birds: (G1) unvaccinated and unchallenged control; (G2) vaccinated on day 0; (G3) challenged on day 1; (G4) vaccinated on day 0 and challenged on day 14; and (G5) challenged on day 14. For 28 days, the clinical signs of infection, weight and feed conversion of the birds, and excretion of oocysts in the feces were evaluated. Macroscopic analysis of intestinal lesions in birds was also performed. After vaccination in G2, G3, and G4 as well as after challenge in G3, G4, and G5, there was an increase in oocyst excretion. In the analysis of weight gain, the difference in final weight between groups G3 and G4 is - 105.74 g per bird. Therefore, if we multiply this value by the average number of birds slaughtered per day in a medium/large slaughterhouse (250,000), we have 26,435 kg of chicken meat per day of slaughter, representing 581,570 kg of monthly losses (22 days of slaughter/month), or approximately R$3,489,420.00 (US$872,355.00), considering the commercial value at R$6.00/kg (US$ 1.5/kg). Thus, the productive and economic impact of coccidiosis in broiler chickens is evident, and the importance of vaccination to prevent the occurrence of the disease and reduce subsequent loss is highlighted.

Detection of anti-Sarcocystis spp. antibodies in domestic cats, in southern Brazil.

Parasites of the genus Sarcocystis can infect several species of animals and cause multiple diseases such as equine protozoal myeloencephalitis. Felines are considered hosts of this protozoa; therefore, the present study aimed to detect anti-Sarcocystis spp.-specific antibodies in domestic cats that were under clinical evaluation, using the indirect immunofluorescence antibody test. Anti-Sarcocystis-specific immunoglobulin Gs were detected in 24 out of 497 (4.82%) cat serum samples. These findings support the fact that natural Sarcocystis infections do occur in cats. Furthermore, it highlights the importance of domestic cats as both intermediate and definitive hosts in the Sarcocystis life cycle, maintaining the parasite and serving as a source of infection for various other animals. To the best of our knowledge, this is the first study to identify antibodies against the genus Sarcocystis in cats from a region in southern Brazil.

Rhipicephalus microplus larvae: first report in bats, Molossus rufus.

Cattle are the main hosts of Rhipicephalus microplus; however, this ixodid can also parasitize other animal species. We collected a specimen of R. microplus larvae from one bat Molossus rufus (Mammalia, Chiroptera) from the Cachoeirinha municipality (29° 56' 52'' S and 51° 5' 43'' W), Rio Grande do Sul, Brazil. The specimen was taxonomically identified using identification keys (Vargas 2006). This study reports the first occurrence of this species parasitizing insectivorous bats in Brazil.

Isolation and characterization of Buchananella hordeovulneris from feline pyothorax.

A 1-year-old female mixed-breed cat was admitted to a veterinary hospital in Southern Brazil with tachypnea, low thoracic amplitude, restrictive breathing pattern, and cyanotic mucous membranes 2 days after elective castration surgery. Radiography revealed pleural effusion, and approximately 100-200 mL of fluid was collected by thoracocentesis. The reddish purulent exudate contained large numbers of yellowish-white granules with branched filamentous structures on cytological examination. The fluid was plated on blood agar and incubated under aerobiosis at 37 °C. On the third day of incubation, circular, dry, and opaque colonies, measuring < 0.5 mm in diameter, were observed. Their phenotypic and molecular characteristics were compatible with Buchananella hordeovulneris (basonym: Actinomyces hordeovulneris), a pathogenic actinomycete rarely detected in cats. Our findings indicate that B. hordeovulneris should be included in the differential diagnosis of pyothorax in cats together with Actinomyces spp. and Nocardia spp. Taxonomic confirmation of disease-causing microorganisms in animals is important to understand the course of infection and its association with disease epidemiology.

Detection of Sarcocystis spp. and Toxoplasma gondii in swine and detection of DNA of these protozoa in tissues and sausages.

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.

Prevalence and molecular detection of Leishmania spp. in bats from Rio Grande do Sul state, Brazil.

This study aimed to detect the occurrence of infection by Leishmania spp.in bats from 34 municipalities of Rio Grande do Sul state (RS; southern Brazil) from 2016 to 2021. A total of 109 bats were provided by the Centro Estadual de Vigilância em Saúde of RS, including six species belonged to Molossidae family, six to Vespertilionidae family, and two to Phyllostomidae family. Leishmania spp. was identified using the nested-PCR method by amplifying the SSU rDNA ribosomal subunit gene into four organ pools: (1) the liver, spleen, and lymph node; (2) heart and lungs; (3) skin; and (4) bone marrow of each bat. Three (3/109, 2.7%) animals tested positive for Leishmania spp. The respective PCR-positive organs came from pools 1 and 3. Two bats (Tadarida brasiliensis) were from the municipality of Canoas, and sequences analysis confirms the species identification as Leishmania infantum. In the third bat (Molossus molossus), from Rio Grande, it was not possible to determine the protozoa species, being considered Leishmania spp. Our results indicate that bats can participate in the biological cycle of Leishmania spp. and perform as host, reservoir, and/or source of infection of the protozoa in different areas of RS. More studies will be needed to elucidate the role of these Chiropteras in the circulation of Leishmania spp. This is the first study reporting the occurrence of Leishmania spp. in bats in Rio Grande do Sul state, southern Brazil.

Frequent Infection of Cats With SARS-CoV-2 Irrespective of Pre-Existing Enzootic Coronavirus Immunity, Brazil 2020.

Carnivores such as cats and minks are highly susceptible to SARS-CoV-2. Brazil is a global COVID-19 hot spot and several cases of human-to-cat transmission have been documented. We investigated the spread of SARS-CoV-2 by testing 547 domestic cats sampled between July-November 2020 from seven states in southern, southeastern, and northeastern Brazil. Moreover, we investigated whether immune responses elicited by enzootic coronaviruses affect SARS-CoV-2 infection in cats. We found infection with significantly higher neutralizing antibody titers against the Gamma variant of concern, endemic in Brazil during 2020, than against an early SARS-CoV-2 B.1 isolate (p<0.0001), validating the use of Gamma for further testing. The overall SARS-CoV-2 seroprevalence in Brazilian cats during late 2020 validated by plaque reduction neutralization test (PRNT90) was 7.3% (95% CI, 5.3-9.8). There was no significant difference in SARS-CoV-2 seroprevalence in cats between Brazilian states, suggesting homogeneous infection levels ranging from 4.6% (95% CI, 2.2-8.4) to 11.4% (95% CI, 6.7-17.4; p=0.4438). Seroprevalence of the prototypic cat coronavirus Feline coronavirus (FCoV) in a PRNT90 was high at 33.3% (95% CI, 24.9-42.5) and seroprevalence of Bovine coronavirus (BCoV) was low at 1.7% (95% CI, 0.2-5.9) in a PRNT90. Neutralizing antibody titers were significantly lower for FCoV than for SARS-CoV-2 (p=0.0001), consistent with relatively more recent infection of cats with SARS-CoV-2. Neither the magnitude of SARS-CoV-2 antibody titers (p=0.6390), nor SARS-CoV-2 infection status were affected by FCoV serostatus (p=0.8863). Our data suggest that pre-existing immunity against enzootic coronaviruses neither prevents, nor enhances SARS-CoV-2 infection in cats. High SARS-CoV-2 seroprevalence already during the first year of the pandemic substantiates frequent infection of domestic cats and raises concerns on potential SARS-CoV-2 mutations escaping human immunity upon spillback.

Macroscopic, histological, and molecular aspects of Sarcocystis spp. infection in tissues of cattle and sheep.

The macroscopic, histological, and molecular aspects of Sarcocystis spp. were examined in the tissues of two cattle and four sheep, 16 and eight fragments analyzed respectively, condemned in the slaughterhouse. All 24 samples were collected and analyzed for detecting macrocysts and macroscopic lesions. Subsequently, subdivided for direct examination, polymerase chain reaction and histopathological examination. All sheep tissues samples had grossly white round to oval tissue cysts, ranging from 0.3 to 1 cm in diameter. In contrast, cattle tissues did not present grossly visible cysts but had randomly distributed white-yellow foci with irregular contours. All samples from cattle and sheep had microscopic cysts. In the histological examination of sheep tissues, circular to elongated, encapsulated, basophilic structures ranging from 30 to 3,000 µm in length and 20 to 1,000 µm in width were observed within the skeletal muscle fibers. In cattle tissues, all cardiac muscle four fragments analyzed contained circular to elongated basophilic structures inside cardiomyocytes and in some Purkinje fibers. PCR were performed using the primers: 2L and 3H. In conclusion, all 24 tissues were infected with Sarcocystis spp., and S. gigantea (in sheep) and S. cruzi (in cattle). were the identified species by sequencing.

Natural Occurring Muscular Sarcocysts in Urban Domestic Cats (Felis catus) Without Sarcocystis-Associated Disease.

PURPOSE: Despite of classically acting as definitive hosts of different Sarcocystis species, domestic cats have been pointed out as possible intermediate hosts of S. neurona and S. felis. Nonetheless, details concerning natural sarcocyst development in cats without Sarcocystis-associated disease are scarce. This study aimed to characterize the natural occurrence of muscular sarcocysts in a random group of cats submitted for necropsy. METHODS: One hundred cats necropsied at a Veterinary Pathology Service were included. Nine different muscular tissues from each cat were sampled for histological analysis and Polymerase Chain Reaction (PCR) using multispecies primers for Sarcocystis neurona, Neospora caninum and Toxoplasma gondii. PCR-positive cases were sequenced for genus and species identification. Epidemiologic data was also analyzed. RESULTS: Tissue sarcocysts were identified in hematoxylin and eosin-stained slides from five cats, and S. neurona was the only confirmed species. Multifocal sarcocysts affecting two or more muscles were common among positive cats (4/5). Sarcocysts were identified within vastus lateralis (3/5), intercostal (3/5), subscapular (2/5) and diaphragm (2/5) sections. These cysts were always incidental necropsy findings. All sarcocyst-positive cats were from urban areas, among which two were feral and three were pets. Outdoor access was consistently reported. Two cats were positive for retrovirosis, and treatments with potentially immunosuppressive drugs were never stated. CONCLUSIONS: This study describes the natural occurrence of S. neurona muscular sarcocysts in a random group of cats without Sarcocystis-associated disease. These findings reinforce the participation of feral and pet cats from urban areas as natural intermediate hosts of S. neurona.

Molecular Detection and Characterization of Sarcocystis Infection in Naturally Infected Buffaloes, Brazil.

ABSTRACT: Sarcocystosis is a disease caused by various Sarcocystis species, a coccidian protozoan parasite that infects humans and animals and is commonly found in ruminants. Although Sarcocystis occurs all over the world, the species responsible for infecting buffaloes in Brazil have not been identified. In this study, we used molecular methods to estimate the prevalence of natural Sarcocystis infection in buffaloes. Phylogenetic analyses were conducted for the first time to identify the species of this protozoan that are responsible for infecting buffalos in southern Brazil, Rio Grande do Sul state. Heart samples from 80 buffaloes were subjected to microscopic examination followed by molecular analysis. Microcysts were present in 19 (23.75%) of 80 samples. The genomic DNA from the 19 cyst samples was extracted and amplified, and six sequences were obtained. The analysis was performed with the StandenPackage software, and the nucleotide sequences generated were analyzed by comparison with sequences in GenBank. All the sequenced samples were identified as Sarcocystis levinei.

Contaminated water confirmed as source of infection by bioassay in an outbreak of toxoplasmosis in South Brazil.

The protozoan Toxoplasma gondii is a causative agent of toxoplasmosis, an important and widespread zoonotic disease. The transmission of this disease in humans includes ingestion of sporulated oocysts present in contaminated water or food. T. gondii oocysts are widely distributed and toxoplasmosis is considered a major food- and waterborne pathogen worldwide, making drinking water containing sporulated T. gondii oocysts a major source of contamination for people. In the first half of 2018, an unprecedented outbreak of toxoplasmosis was reported in the city of Santa Maria, southern Brazil. The temporal and spatial distribution of the cases strongly suggested a waterborne infection. Thus, the aim of this study was to investigate a possible involvement of treated water as a source of the outbreak. For this, piglets received potentially contaminated water ad libitum for 21 days and the infection was monitored by serology through IFAT and investigation of T. gondii DNA in tissues by PCR amplification of a 529 bp followed by mouse bioassays. All piglets receiving test water ad libitum for 21 days as well as positive controls seroconverted to T. gondii. T. gondii DNA was detected in 62.5% of the piglets that received test water. All mice inoculated with tissues from each positive piglet were PCR-positive. These results strongly indicated the presence of viable oocysts in the test water administered to the animals during the study.