Glanders Diagnosis in an Asymptomatic Mare from Brazil: Insights from Serology, Microbiological Culture, Mass Spectrometry, and Genome Sequencing.

This manuscript elucidates the occurrence of glanders in an asymptomatic mare from Brazil presenting positive Burkholderia mallei antibody titers. The diagnosis was established through a multi-pronged approach encompassing microbiological culture, mass spectrometry, and genome sequencing. The outbreak occurred in 2019 in Tatuí, São Paulo, Brazil, and the infected mare, despite displaying no clinical symptoms, had multiple miliary lesions in the liver, as well as intense catarrhal discharge in the trachea. Samples were collected from various organs and subjected to bacterial isolation, molecular detection, and identification. The strain was identified as B. mallei using PCR and confirmed by MALDI-TOF mass spectrometry. Whole-genome sequencing revealed a genome size of 5.51 Mb with a GC content of 65.8%, 5871 genes (including 4 rRNA and 53 tRNA genes), and 5583 coding DNA sequences (CDSs). Additionally, 227 predicted pseudogenes were detected. In silico analysis of different genomic loci that allow for differentiation with Burkholderia pseudomallei confirmed the identity of the isolate as B. mallei, in addition to the characteristic genome size. The BAC 86/19 strain was identified as lineage 3, sublineage 2, which includes other strains from Brazil, India, and Iran. The genome sequencing of this strain provides valuable information that can be used to better understand the pathogen and its epidemiology, as well as to develop diagnostic tools for glanders.

From weaning to breeding age: Presence of antibodies to Toxoplasma gondii in sheep in the state of Espírito Santo, southeastern Brazil.

Sheep are intermediate hosts of the protozoon Toxoplasma gondii, a zoonotic parasite that that may cause abortion and reproductive losses in this species. Previous studies have shown that its prevalence is high in sheep herds in Brazil, but no information from the state of Espírito Santo was available. The aim of this study was to improve the epidemiological data in different regions of this state by analyzing occurrences of anti-T. gondii antibodies in young female sheep, before reproductive age, using IFAT (cutoff ≥64). In addition, variables relating to these animals and farm management were investigated for significance of associations. A total of 312 sheep, aged 3 to 12 months, on five farms in five municipalities, were sampled. Among these 312 animals, 135 (43.26%) were reactive, and 70 positive sheep (51.8%) presented high titers (more than three times the cutoff).Eight-month-old sheep presented the lowest occurrence and 7 and 11-month-old sheep, the highest. At least one animal was positive in each municipality, thus confirming the endemic status of toxoplasmosis in sheep in this region. None of the variables analyzed, relating to animals and herds, presented any association with T. gondii occurrence (p > 0.05). The number of females that were close to sexual maturity (12 months) and which were reactive to T. gondii was less than 50%. These ewes would probably be more susceptible to reproductive problems.

Seroprevalence and incidence of Toxoplasma gondii and Neospora caninum infection in naturally exposed domestic dogs from a rural area of São Paulo state, Brazil.

Dogs are hosts of the protozoans Toxoplasma gondii, which causes an important public health disease, and Neospora caninum. Studies that have evaluated toxoplasmosis and neosporosis for prolonged periods in dog populations are rare. We analyzed infection by both parasites in a domestic dog population over three consecutive years in São Paulo state, Brazil. In the 1st, 2nd and 3rd years of collection, 181, 193 and 172 domiciles were visited, and blood samples of 331, 371 and 348 dogs were collected for antibody serology, respectively. The seroprevalence of T. gondii in each year was 27.2%, 22.5% and 43.9%, respectively, and that of N. caninum was 7.8%, 4.8% and 6.8%, respectively. The incidence rates for T. gondii in the 2nd and 3rd collections were 13.2% and 30.0%, and those for N. caninum were 3.3% and 4.4%, respectively. Positive and negative serological conversions for both agents occurred at high frequencies during the study period. This study reveals the canine population's serological profile and demonstrates the constant exposure of dogs to the investigated pathogens, indicating the need for prevention and control measures in the region.

Systematic monitoring of glanders-infected horses by complement fixation test, bacterial isolation, and PCR.

Glanders is an equine zoonosis caused by Burkholderia mallei that is responsible for considerable economic loss. Complement fixation testing (CFT) using warm or cold incubation are recommended by the OIE, but many routinely used detection tests may present misleading results. To increase accuracy of glanders diagnosis and establish an appropriate protocol in collaboration with the National Equine Health Program, seven horses positive for glanders kept in isolation in Brazil were examined fortnightly by CFT, microbiological screening, and molecular testing. Warm and cold serologies with USDA and c.c.Pro antigens, respectively, were performed on 132 samples using the US Department of Agriculture protocol. The warm and cold serologies showed, respectively,12.9% and 17.3% seroreactive, 85.7% and 65.2% non-reactive, 0.8% and 3% inconclusive, and 0% and 2.3% anticomplementary. The agreement of CFT protocols was moderate. Of 213 clinical samples submitted to selective culture (167 nasal swabs, 5 ocular swabs, 3 lymph node punctures, and 38 tissue samples from four horses that died), 1.9% tested positive for B. mallei. Fourteen samples and one nasal swab (7%) tested positive with PCR. Cold CFT with the USDA and c.c.Pro antigens, in combination with PCR to increase sensitivity, may be useful for diagnosis of chronic glanders.

Epidemiological study of Giardia duodenalis infection in companion dogs from the metropolitan area of São Paulo Brazil.

Giardia duodenalis is a zoonotic pathogen associated with gastrointestinal disease that has a direct life cycle, with cysts eliminated in the faeces of an infected host being ingested by a susceptible host. In Brazil, studies of chronically infected adult dogs estimated a prevalence of 10%-20%. Diagnosis of giardiasis, as a cause of diarrhoea is important for the global One-Health guidelines when controlling cyst dissemination in the environment. We investigated the prevalence of G. duodenalis in the pet dog population of the metropolitan area of Sao Paulo, compared the efficacy of direct tests available to the veterinary clinical practice and attempted to identify possible risk factors associated with the parasite. Ten veterinary practices distributed throughout the municipality randomly performed the rapid SNAP ELISA test on canine faecal samples, and dog owners provided information specific to the animal via a questionnaire. The samples were also analysed using sucrose and zinc sulphate flotation techniques. Sensitivity and specificity of the tests were used to calculate required number of samples and true prevalence. Significance, agreement among tests, and odds ratio (OR) were assessed with a confidence interval (CI) of 95%. The prevalence of G. duodenalis in dogs (n = 265) was 6.9% (CI 3.47-11.21). Positive tests were significantly more frequent in animals younger than 1 year, with an OR for G. duodenalis occurrence nearly 7-fold that of older dogs. Direct diagnosis tests showed high agreement (96.1%, κ = 0.729; p < .0001) showing that the combined techniques provide a highly accurate diagnosis. Results indicated that the control of the pathogen has been improving in the pet dog population in metropolitan Sao Paulo, but management tools including diagnosis, immunization, and treatment, especially in puppies, must be continued in order to advance towards continuous decrease of the disease.

Seroprevalence and incidence of Toxoplasma gondii and Neospora caninum infection in naturally exposed domestic dogs from a rural area of São Paulo state, Brazil / Soroprevalência e incidência de infecção por Toxoplasma gondii e Neospora caninum em cães domésticos naturalmente expostos em uma área rural no estado de São Paulo, Brasil

Abstract Dogs are hosts of the protozoans Toxoplasma gondii, which causes an important public health disease, and Neospora caninum. Studies that have evaluated toxoplasmosis and neosporosis for prolonged periods in dog populations are rare. We analyzed infection by both parasites in a domestic dog population over three consecutive years in São Paulo state, Brazil. In the 1st, 2nd and 3rd years of collection, 181, 193 and 172 domiciles were visited, and blood samples of 331, 371 and 348 dogs were collected for antibody serology, respectively. The seroprevalence of T. gondii in each year was 27.2%, 22.5% and 43.9%, respectively, and that of N. caninum was 7.8%, 4.8% and 6.8%, respectively. The incidence rates for T. gondii in the 2nd and 3rd collections were 13.2% and 30.0%, and those for N. caninum were 3.3% and 4.4%, respectively. Positive and negative serological conversions for both agents occurred at high frequencies during the study period. This study reveals the canine population's serological profile and demonstrates the constant exposure of dogs to the investigated pathogens, indicating the need for prevention and control measures in the region.

Resumo Os cães são hospedeiros dos protozoários Toxoplasma gondii, que causam uma importante doença para a saúde pública, e Neospora caninum. Estudos que avaliam a toxoplasmose e a neosporose por períodos prolongados em populações caninas são raros. Foi analisada a infecção por esses dois parasitas em uma população de cães domésticos e domiciliados por três anos consecutivos, no Estado de São Paulo, Brasil. Nos 1º, 2º e 3º anos de coletas, 181, 193 e 172 domicílios foram visitados, nos quais foram coletadas amostras de sangue de 331, 371 e 348 cães para sorologia, respectivamente. A soroprevalência de T. gondii em cada ano foi de 27,2%, 22,5% e 43,9%, respectivamente; e a de N. caninum foi de 7,8%, 4,8% e 6,8%, respectivamente. As taxas de incidência para T. gondii, nas 2ª e 3ª coletas, foram de 13,2% e 30,0%, respectivamente; e para N. caninum, 3,3% e 4,4%, respectivamente. As conversões sorológicas positivas e negativas para ambos os agentes ocorreram em alta frequência durante o período analisado. Este estudo revela o perfil sorológico da população canina e demonstra sua constante exposição aos patógenos investigados, o que requer medidas de prevenção e controle na região.

Congenital Transmission of Toxoplasma gondii After Experimental Reinfection With Brazilian Typical Strains in Chronically Infected Sheep.

Toxoplasma gondii is a cause of congenital diseases, miscarriages and stillbirths in production animals. In Brazil, non-archetypal genotypes of the parasite may be related to severe disease. Experimental infection with T. gondii was studied in sheep to analyse congenital transmission-related parameters in reinfections with different Brazilian parasite strains. Thirteen T. gondii-seronegative sheep were orally infected with 2 × 103oocysts for the primary infection: G1 (4 animals) were inoculated with TgCatBr71 strain (Type BrI genotype) and G2 andG3 (5 and 4 animals, respectively) withTgCatBr60 strain (Type BrIII genotype). After chronification of infection, the animals were impregnated. A second infection was performed after 60 days of gestation. TheG1 andG3 animals were inoculated withTgCatBr60BrIII and the G2 animals withTgCatBr71 BrI oocysts. The effects of reinfection were compared with a control group (5 animals) through physical examination, ultrasound imaging and serology. Ovine experimental infections were evaluated using mouse bioassays, molecular analysis, serological tests, histopathology, and immunohistochemistry. No abortions occurred; a seropositive lamb and a mummified fetus from G2-BrIIIxBrI were produced. The vertical transmission rate detected in lambs from chronically infected sheep was 31.6% (6/19). It is demonstrated that reinfection and subsequent congenital transmission occured in one sheep with a primary Brl infection challenged with BrIII genotype of T. gondii. In a twin pregnancy from G2-BrIIIxBrI, congenital transmission from a latent infection was detected in both lambs. Congenital transmission could not be tracked in three lambs. Overall, previous T. gondii infection may fail to protect against congenital transmission from a reinfection and primary infection induced insufficient protection against vertical transmission which must be taken into account in decision-making for the use of seropositive animals as breeders. Similar trials with larger groups and contemplating host cellular immune response studies should be conducted to evaluate the actual impact of T. gondii reinfection involving different strains in sheep.

Experimental Neospora caninum infection in chickens (Gallus gallus domesticus) with oocysts and tachyzoites of two recent isolates reveals resistance to infection.

The importance of birds in the biological cycle of Neospora caninum is not clear. We report unsuccessful Neospora infection in chickens (Gallus gallus domesticus) using two isolates of N. caninum. In experiment #1, 30 White Leghorn chickens were orally inoculated with viable N. caninum oocysts (NC-SP1 isolate, 200 oocysts per bird) via the crop at 21days of age. Groups of three birds were euthanised at intervals of 7days (a total of 9weeks) and one group was challenged with the same oocyst dose at 37daysp.i. and observed for 11weeks. Blood samples were collected weekly, and sera were tested using IFAT. Chicken tissues were collected for PCR, quantitative PCR and immunohistochemistry. Two dogs approximately 45days of age were fed with tissues from chickens euthanised at 138 and 159daysp.i. The results indicated that the chickens were resistant to neosporosis as revealed by failure to seroconvert, to detect parasite DNA or N. caninum antigen by immunohistochemistry in inoculated bird tissues, and by no oocyst excretion by the dogs fed avian tissues. Similar results were obtained in experiment #2, in which 34 1-week-old chickens were each s.c. inoculated with 100,000 tachyzoites of the NcWTDMn1 isolate of N. caninum. The chickens were euthanised on days 7, 15, 22, 28, 36 and 60p.i. At necropsy, all tissues and serum from each bird were collected. All chickens remained asymptomatic, and N. caninum antigen was not detected by immunohistochemistry. Seven chickens euthanised at day 60p.i. demonstrated low (1:25 dilution) levels of antibodies by using the Neospora agglutination test. Two 12-week-old dogs fed tissues pooled from 10 inoculated chickens euthanised at day 60p.i. did not excrete N. caninum oocysts. This investigation indicates that chickens are resistant to experimental infection by N. caninum.

Isolation and biological and molecular characterization of Neospora caninum (NC-SP1) from a naturally infected adult asymptomatic cattle (Bos taurus) in the state of São Paulo, Brazil.

The biological and genetic diversity of Neospora caninum is very limited because of availability of only a few viable isolates worldwide. This study describes the isolation and biological and molecular characterization of a new viable isolate of N. caninum (NC-SP1), from a cattle in Brazil. Approximately 400 g of brain from a naturally infected adult male cattle from an abattoir was fed to a 2-month-old dog. Neospora-like oocysts were observed on day 7 post-inoculation (PI) and the duration of oocyst shedding was 14 days. The DNA obtained from oocysts was characterized molecularly and the final sequence was 99% identical to homologous sequences of N. caninum available in GenBank®. For bioassay, gerbils (Meriones unguiculatus) were orally inoculated with 10 100 and 1000 oocysts; all gerbils remained clinically normal but developed N. caninum antibodies 14 days PI. Cell culture isolation was successful using the brain homogenate from one of the gerbils and tachyzoites were observed 24 days PI. Microsatellite genotyping revealed a unique genetic profile for this new reference isolate.

Infection of mice with oocysts of Toxoplasma gondii by oral route showed differences of virulence from Brazilian RFLP genotypes BrI and BrIII.

South American strains of Toxoplasma gondii present higher genetic diversity than classical European strains. We compared the virulence of two non-archetypal Brazilian genotypes of T. gondii to mice. Oocysts of four isolates, two genotype BrI (TgCatBr71 and TgShBr11) and two BrIII (TgCatBr74 and TgCatBr60) were obtained from cats fed experimentally infected mice. After sporulation, 5.0×10(1) and 1.0×10(2) oocysts were orally administrated to Swiss albine mice in Experiments #1 and #2, respectively (4-10 mice/group). Humoral response from dead and surviving mice was analyzed on days 9 to 35 post-infection. Microscopic observations of lungs and brains were performed for tachyzoites and cysts visualization in fresh preparations. Negative results were tested by PCR. Virulence after infection with oocysts is dose dependent for genotype BrIII isolates, but not for BrI. Differences in mortality were observed among isolates from genotype BrIII on Experiment #1. Intra-genotype phenotypic variation related to the parasite stage of infection was demonstrated and this characteristic should be further studied and may influence future work regarding the role of virulence amid hosts.

F200Y polymorphism in the ß-tubulin gene in field isolates of Haemonchus contortus and risk factors of sheep flock management practices related to anthelmintic resistance.

Haemonchus contortus is the most prevalent and pathogenic nematode of sheep in tropical areas. The objectives of this study were to assess the frequency of the F200Y polymorphism on the ß-tubulin gene in third-stage larvae of H. contortus from 33 sheep flocks in São Paulo state, Brazil, and to associate this frequency to risk factors based on farm management practices. The resistance allele frequency varied from 9 to 74%, and the resistance genotype frequency varied from 0 to 66.7%. Resistance genotype frequencies higher than 40% were associated with multiple risk factors - new sheep farming enterprises, the absence of farm records, the use of Dorper and Suffolk breeds, rotational grazing, the lack of wetlands on farms, pasture sharing with cattle or horses, frequent incorporation of animals into the flock, semi-intensive farming systems, whole-flock treatment, failure to use the FAMACHA method, lack of the dose-and-move practice, anthelmintic rotation after each application, visual estimation of animal weight for treatment, and lack of drug combination use. It can be concluded that genotyping the F200Y polymorphism can be used to monitor the resistance in sheep flocks and the knowledge of management strategies at the farm level is important to identify drug resistance related factors.

Multidrug and multispecies resistance in sheep flocks from São Paulo state, Brazil.

The economic importance of sheep production is increasing worldwide simultaneously with the emergence of parasitic resistance. This study aimed to survey the current situation of management practices and parasite resistance in sheep flocks in São Paulo state, Brazil. A questionnaire was given to 35 sheep farmers to obtain information related to flock management practices. Of these flocks, 30 were submitted to the fecal egg count reduction test (FECRT) with at least one of the five following anthelmintics: albendazole, closantel, ivermectin, levamisole, and moxidectin, for comparison against an untreated control group. In the survey, the median number animals per flock was 301, mainly of the Santa Ines breed (in 75.8% of the flocks) and crossbred animals (in 54.5% of the flocks). The predominant farming system was semi-intensive (82.9%), using rotational grazing (80%). Selective treatment was based on FAMACHA grade (47.1%) and in clinical signs (41.2%). The most often applied anthelmintics were macrocyclic lactones (42.9-54.2% in the last three applications). Considering the anthelmintics employed in this study, 10.7% of the farms' flocks were resistant to three, 35.7% to four, and 53.6% to all five anthelmintics. The main helminth genera observed before and after treatments were Haemonchus sp. (75.8%) and Trichostrongylus sp. (19.1%), but all observed genera (Cooperia sp., Oesophagostomum sp., and Strongyloides sp.) were detected by the FECRT. Considering efficacy values less than or equal to 90% in the FECRT as resistant, 100% of flocks were resistant to albendazole and ivermectin, 96.6% to moxidectin, 92.9% to closantel, and 53.6% to levamisole. It is thus possible to conclude that multidrug resistance is widespread in sheep flocks in São Paulo state, Brazil, and this involves all prevalent helminth genera.