Detection of Trypanosoma cruzi DNA in lizards: Using non-lethal sampling techniques in a sylvatic species with zoonotic reservoir potential in Chile.

Several reptile species have been described as hosts of Trypanosoma cruzi, the causative agent of Chagas disease, and therefore, they have become vertebrates of epidemiological interest. In recent decades, there has been a growing interest in animal welfare, especially in populations with small numbers where lethal sampling could have catastrophic consequences, and non-lethal methodologies have been developed for detecting zoonotic parasites. In this study, we compared three non-lethal sampling methodologies for detecting T. cruzi DNA in 21 captured specimens of the native lizard Liolaemus monticola, collected from the semiarid Mediterranean ecosystem of Chile. Specimens were subjected to xenodiagnosis (XD), tail clipping, and living syringe sampling procedures to evaluate whether lizards could serve as sentinel species for T. cruzi in endemic regions. To detect the protozoan, real-time PCR (qPCR) was performed on the DNA extracted from the samples (intestinal contents, tail tissues, and blood from living syringes). Trypanosoma cruzi DNA was detected in 12 of 21 lizards, considering all three methodologies. By XD, 12 specimens showed infection (57.1 %), and both living syringe and tail sampling methodologies detected only one infected lizard (4.8 %). Therefore, T. cruzi can be detected in lizards by qPCR using the three methodologies but XD is by far the most effective non-lethal detection methodology. The use of tail and living syringe methodologies showed a large underestimation; however, they might be options for monitoring the presence of T. cruzi in lizard populations when large sample sizes are available.

Immunostaining and gene expression of epidermal growth factor receptors (HER1/HER2) in canine cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a neoplasm type often diagnosed in dogs. However, studies focused on further investigating its molecular biology, mainly biomarkers to help implementing new therapies, remain scare in the literature. Thus, immunostaining and the gene expression of epidermal growth factor receptors (HER1 and HER2) in canine cSCC presenting different cell differentiation degrees were herein assessed. Thirty-two (32) canine cSCC were selected, classified based on to their cell differentiation degree and subjected to immunohistochemical study to assess HER1 and HER2 immunostaining intensity and distribution. In addition, HER1 and HER2 gene expression was investigated through real-time PCR. Membranous and cytoplasmic immunostaining were observed in both markers. HER2 prevailed in poorly differentiated cSCC; there was positive protein expression correlation between both markers. Mean HER1 gene expression was higher in moderately differentiated, whereas mean HER2 gene expression was higher in poorly differentiated cSCC. Moreover, there was gene expression correlation between markers, regardless of cell differentiation degree. Thus, HER2 protein immunostaining and gene expression were higher in poorly differentiated canine cSCC and it enabled understanding that increase observed in this epidermal growth factor receptor is proportional to this neoplasm's cell differentiation degree in canine species. Results in the current study helped better understanding canine cSCC's molecular biology; however, it is relevant studying other markers aiming to investigate signaling pathways.

Evaluation of nasal swab and nasal wipe for detection of Influenza A in swine using Bayesian latent class analysis.

Influenza A virus (IAV) is an important pathogen in Brazilian swine herds, and monitoring the viral circulation is essential to control and reduce the transmission. Surveillance programs for IAV are often based on individual piglets level sampling, making the evaluation of the available diagnostic tools crucial to assessing IAV circulation in herds. Thus, two sample collection methodologies were compared in pig herds in southern Brazil to detect IAV by RT-qPCR: nasal swab (NS) and nasal wipe (NW). A Bayesian latent class model (BLCM) was set for two tests and two populations. The NW and NS used are more specific (higher than 95 % for both) than sensitive. The sensitivity for NW was lower than the NS, 84.14 % (70 % - 95 %; posterior probability interval (PPI): 95 %) and 87.15 % (73 % - 97 %; PPI: 95 %), respectively, and the specificity was 95 % (90 % - 99 %; PPI: 95 %) and 99 % (96 % - 100 %; PPI: 95 %), respectively. Although the wipe sample collection loses both sensitivity and specificity compared with nasal swab, differences in test performance were very limited and PPIs largely overlapped. Therefore NW can also be considered a valuable tool. The decision about the use of both techniques should be based on the trade-off between their performance limitations and feasibility in routine monitoring.

Prevalence of Leishmania infection in 205 cats from a referral hospital population in Brazil (2021-2022).

Leishmaniases are a group of neglected diseases of significant public health concern, with Brazil being the primary focus of this disease in the Americas. The municipality of Sobral, in the state of Ceará, is a historical focus of visceral leishmaniasis in both humans and dogs, but data on Leishmania spp. infections in cats are limited. Between April 2021 and February 2022, 205 cats from a referral hospital population were sampled and tested for Leishmania spp. by real-time PCR. Eight cats (3.9%; 95% CI: 1.7-7.5%) tested positive. Among these, three (37.5%) displayed clinical signs compatible with feline leishmaniosis. Non-domiciled cats showed significantly higher positivity compared to domiciled ones (Fisher's exact test, P = 0.0124). Considering their potential role as reservoirs of L. infantum, it is crucial to conduct further studies to understand the Leishmania spp. circulating among cats in Sobral and to implement measures for reducing their exposure to phlebotomine sand fly vectors in this important focus of leishmaniases.

Evaluating the circulation of Ehrlichia canis and Rickettsia spp. in domestic dogs from a semiarid region in Brazil.

Tick-borne diseases are important for animal and human health, because they can cause death if not diagnosed and treated early. Canine monocytic ehrlichiosis (CME) can cause high morbidity in dog populations. Rocky Mountain Spotted Fever (RMSF) is among the most virulent infectious in humans; dogs are also susceptible to infection. The aims of this study were to evaluate the presence of Ehrlichia canis and Rickettsia spp. infections in domestic dogs, and to identify tick species parasitizing dogs among urban areas of two municipalities (Sobral and Alcântaras) in the Ceará State, Northeastern Brazil. A total of 208 domiciled dogs was sampled. After clinical evaluation, blood samples and ticks were collected and submitted to Real-Time Polymerase Chain Reaction (RT-PCR) targeting E. canis DNA. Serum samples were screened by Indirect Immunofluorescence Assays (IFA) for antibodies against different strains of Rickettsia spp. previously recognized in Brazil. The results of this study indicate the molecular detection of E. canis in the state of Ceará, Brazil, where the proportion of canine infection in Sobral (9.9%) was higher than in Alcântaras (5.6%). Rhipicephalus sanguineus sensu lato was the prevalent tick species infesting the dogs in both municipalities (43.5 and 53.3%, respectively). Our serological results indicate that dogs of the study area were at low risk of exposure to these tick-borne Rickettsia spp. of the spotted fever group. Our study offers epidemiological data of these diseases to better understanding Rickettsiales epidemic and enzootic cycles in the Brazilian semiarid region, improving prevention and control measures.

Pathogenic bacteria prevalence in cultured Nile tilapia in Southwest Mexico: A real-time PCR analysis.

The present study investigates molecular-based PCR techniques to estimate the prevalence of fish pathogens in southwest Mexico where recurrent mortality in the tilapia cultures has been observed. Sample of internal organs and lesions of Nile tilapia were taken and analysed in 2018, 2019, 2020 and 2022 to detect bacterial pathogens using PCR. No samples were taken in 2021 due to the COVID-19 pandemic. The real-time PCR conditions were optimized to allow a qualitative reliable detection of the bacteria from fixed fish tissue. A total of 599 pond- and cage-cultured tilapia from the southwestern Mexican Pacific (Guerrero, Oaxaca and Chiapas states) were analysed. In this tropical region, during 2018 and 2019 water temperatures of the tilapia cultures were generally with the optimal range to grow Nile tilapia, although extreme values were recorded on some farms. Most of the tilapia sampled were apparently healthy. No Francisella sp. was detected in any sample, and Staphylococcus sp. was the most prevalent (from 0% to 64%) bacteria from the three states over time. Low prevalence of Aeromonas sp. was found, from 0% to 4.3%, although the fish pathogen Aeromonas dhakensis was not detected. Sterptococcus iniae was only detected in Chiapas in 2019 at a low prevalence (1.4%), while the major tilapia pathogen S. agalactiae was detected at a high prevalence (from 0% to 59%) in the three Mexican states. This is the first detection of these pathogenic bacteria in rural farms using real-time PCR and constitutes a great risk for tilapia aquaculture in Mexico, as well as a potential dispersion of these pathogens to other aquaculture areas.

Histopathological, immunohistochemical and molecular analyses of foetuses from a female dog naturally infected by Leishmania (Leishmania) infantum - Case report.

This report presents anatomo-histopathological, immunohistochemical and molecular analyses of an asymptomatic female dog naturally infected with Leishmania (Leishmania) infantum and her four pups. In addition to routine serological tests, the mother's positivity was confirmed by real-time PCR of a bone marrow sample; the placenta and ovaries, however, were negative with the same analysis. The pups presented no positivity in any of the techniques used. It is suggested that the gestational period and factors related to the female's immunological condition cause greater variability in the occurrence of vertical infection.

Molecular evidence of Bartonella spp. in wild lowland tapirs (Tapirus terrestris), the largest land mammals in Brazil.

The genus Bartonella (Hyphomicrobiales: Bartonellaceae) encompasses facultative intracellular α-proteobacteria that parasite erythrocytes and endothelial cells from a wide range of vertebrate hosts and can cause disease in animals and humans. Considering the large diversity of vertebrate species that may act as reservoirs and arthropod species that may be associated with Bartonella transmission, the exposure of animals and humans to these microorganisms is likely underestimated. The present study aimed to investigate the occurrence of Bartonella sp. in wild tapirs (Tapirus terrestris; Perissodactyla: Tapiridae) from two biomes in Brazil: Pantanal and Cerrado. Ninety-nine GPS-monitored wild tapirs were sampled in Pantanal (n = 61/99) and Cerrado (n = 38/99). A qPCR (quantitative real-time polymerase chain reaction) assay targeting the nuoG gene was used for the screening for Bartonella spp. DNA. Positive samples were additionally subjected to conventional PCR assays targeting five molecular markers (ribC, gltA, rpoB, groEL, ITS). Eight (8/99; 08,08%) animals were positive in the qPCR assay for Bartonella spp.: 7 from Cerrado (7/8; 87.5%) and 1 from Pantanal (1/8; 12.5%). The 5 Bartonella ribC sequences obtained from tapirs' blood samples grouped together with Bartonella henselae obtained from cats, humans, wild felids and Ctenocephalides felis (Siphonaptera: Pulicidae) fleas. To the best of author's knowledge, this is the first report of Bartonella sp. in Tapirus terrestris. This finding contributes to the understanding of the occurrence of B henselae in wild mammals from Brazil as well as expands the knowledge regarding the potential vector-borne pathogens that may affect wild tapis from Cerrado and Pantanal biomes.

Molecular identification of Bartonella spp. and Rickettsia felis in fox fleas, Chile.

Seventy-five flea pools (one to ten fleas per pool) from 51 Andean foxes (Lycalopex culpaeus) and five South American grey foxes or chillas (Lycalopex griseus) from the Mediterranean region of Chile were analyzed for the presence of DNA of Bartonella spp. and Rickettsia spp. through quantitative real-time PCR for the nouG and gltA genes, respectively. Positive samples were further characterized by conventional PCR protocols, targeting gltA and ITS genes for Bartonella, and gltA, ompA, and ompB genes for Rickettsia. Bartonella was detected in 48 % of the Pulex irritans pools (B. rochalimae in three pools, B. berkhoffii in two pools, B. henselae in one pool), and 8 % of the Ctenocephalides felis felis pools (B. rochalimae, one pool). Rickettsia was confirmed in 11 % of P. irritans pools and 92 % of the Ct. felis pools. Characterization confirmed R. felis in all sequenced Rickettsia-positive pools. All Ct. canis pools were negative. A Ct. felis pool from a wild-found domestic ferret (Mustela putorius furo) also resulted positive for R. felis. Although opportunistic, this survey provides the first description of zoonotic pathogens naturally circulating in fleas parasitizing Chilean free-living carnivores.

MicroRNA qPCR normalization in Nile tilapia (Oreochromis niloticus): Effects of acute cold stress on potential reference targets.

The Nile tilapia (Oreochromis niloticus) is one of the most important cultured fish worldwide, but tilapia culture is largely affected by low temperatures. Recent studies suggest that microRNAs (miRNAs) regulate cold tolerance traits in fish. In general, qPCR-based methods are the simplest and most accurate forms of miRNA quantification. However, qPCR data heavily depends on appropriate normalization. Therefore, the aim of the present study is to determine whether the expression of previously tested, stably expressed miRNAs are affected by acute cold stress in Nile tilapia. For this purpose, one small nuclear RNA (U6) and six candidate reference miRNAs (miR-23a, miR-25-3, Let-7a, miR-103, miR-99-5, and miR-455) were evaluated in four tissues (blood, brain, liver, and gills) under two experimental conditions (acute cold stress and control) in O. niloticus. The stability of the expression of each candidate reference miRNA was analyzed by four independent methods (the delta Ct method, geNorm, NormFinder, and BestKeeper). Further, consensual comprehensive ranking of stability was built with RefFinder. Overall, miR-103 was the most stable reference miRNA in this study, and miR-103 and Let-7a were the best combination of reference targets. Equally important, Let-7a, miR-23a, and miR-25-3 remained consistently stable across different tissues and experimental groups. Considering all variables, U6, miR-99-5, and miR-455 were the least stable candidates under acute cold stress. Most important, suitable reference miRNAs were validated in O. niloticus, facilitating further accurate miRNA quantification in this species.

Molecular detection and species identification of dermatophytes by SYBR-Green real-time PCR in-house methodology using hair samples obtained from dogs and cats.

The classical dermatophytes diagnosis is based on mycological culture and microscopy observation both human and animal hair, skin, and nail samples. The aim of this work was to develop the new in-house real-time PCR with pan-dematophyte reaction for detection and identification of the main dermatophytes directly from hair samples, providing a simple and rapid diagnosis of dermatophytosis in dogs and cats. An in-house SYBR-Green real-time PCR was designed and used for detecting a DNA fragment encoding chitin synthase 1 (CHS1). A total of 287 samples were processed by culture, microscopic examination with KOH 10%, and real-time PCR (qPCR) analysis. Melting curve analysis of the CHS1 fragment revealed to be reproducible, showing a single distinct peak for each species of dermatophyte, namely Trichophyton mentagrophytes, T. verrucosum, Microsporum canis, and Nannizzia gypsea (formerly M. gypseum). Then, out of the 287 clinically suspected cases of dermatophytosis, 50% were positive for dermatophytes by qPCR, 44% by mycological culture, and 25% by microscopic examination. Microsporum canis was identified in 117 samples tested by culture and 134 samples tested by qPCR, followed by N. gypsea in 5 samples (either tested by culture or qPCR) and T. mentagrophytes detected in 4 and 5 samples when tested by culture or qPCR, respectively. Overall, qPCR allowed the diagnosis of dermatophytosis in clinical samples. The results suggest this newly proposed in-house real-time PCR assay can be used as alternative diagnosis and rapid identification of dermatophytes frequently associated to clinical hair samples of dogs and cats.

The aim of this work was to develop a molecular detection strategy for dermatophytes by SYBR-Green real-time PCR of hair samples from animals. The melting curve analysis of the CHS1 fragment revealed to be reproducible, showing a single distinct peak for distinct dermatophyte species and allowed the diagnosis of dermatophytosis in dogs and cats caused mainly by Trichophyton mentagrophytes, Microsporum sp., and Nannizzia gypsea).

Real-time PCR quantification and histopathological findings of Mycoplasma hyopneumoniae infection in the lungs of pigs slaughtered in São Luís, Maranhão, Brazil / Quantificação por PCR em tempo real e achados histopatológicos da infecção por Mycoplasma hyopneumoniae em pulmão de suínos abatidos na cidade de São Luís, Maranhão, Brasil

Porcine enzootic pneumonia (PES), mainly caused by the bacteria Mycoplasma hyopneumoniae, is the main cause of respiratory problems in pigs. Infection by M. hyopneumoniae leads to production losses and the predisposition of affected animals to secondary infections, which may result in the condemnation of carcasses and organs due to lung lesions at the time of slaughter. The objective of the research was to evaluate the infection by M. hyopneumoniae in pigs submitted to slaughter in São Luís Island/MA, using molecular and histopathological diagnostic methods. One hundred fifty lung samples were collected from inspected (n=65) and non-inspected (n=85) slaughter pigs on São Luís Island, Maranhão, from July 2019 to August 2021. Of the 150 DNA samples collected, 121 showed an amplified product for Cyt B in the PCR assay. Thus, 121 samples were submitted to qPCR of M. hyopneumoniae, of which 44 (36.36%) showed positive results. The mean amount of bacterial load ranged from 1.20 × 101 to 7.20 × 104, with a mean of 1.73 × 104 copies. Of the reagent samples, 81.81% (36 samples) were obtained from non-inspected slaughter, while 18.18% (8 samples) were obtained from slaughterhouses. In the histopathological analysis, 44 positive qPCR samples were evaluated, of which 28 (63.63%) presented results compatible with the main inflammatory process associated with the presence of M. hyopneumoniae, that is, bronchial-associated lymphoid tissue hyperplasia (BALT). Three samples that showed the highest bacterial load (qPCR: 5.63 × 10³, 2.19 × 104 and 7.23 × 104) showed more evident lesions in this study. The microscopic findings associated with the quantifications indicated a relationship between the amount of bacterial load and the presence of microscopic lesions; higher bacterial load in lung tissue is associated with increased histopathologic staining for BALT hyperplasia. In conclusion, the results point to the circulation of the etiological agent in the sampled animals and the need for preventive measures on pig farms in Maranhão with the involvement of producers, sanitary defense and inspection agencies.

A pneumonia enzoótica suína (PES), causada principalmente pela bactéria Mycoplasma hyopneumoniae, é a principal causa de problemas respiratórios em suínos. A infecção por M. hyopneumoniae leva a perdas produtivas e a predisposição dos animais acometidos a infecções secundárias, o que pode resultar em condenação de carcaças e órgãos por lesões pulmonares no momento do abate. O objetivo da pesquisa foi avaliar a infecção por M. hyopneumoniae em suínos submetidos ao abate na Ilha de São Luís, por meio de métodos diagnósticos moleculares e histopatológicos. Para isso, foram coletadas 150 amostras de pulmão de suínos de abate inspecionado (n=65) e não inspecionado (n=85) na Ilha de São Luís/Maranhão, no período de julho de 2019 a agosto de 2021. Das 150 amostras de DNA coletadas, 121 apresentaram produto amplificado para Cyt B no ensaio de PCR. Assim, 121 amostras foram submetidas à qPCR de M. hyopneumoniae, das quais 44 (36,36%) apresentaram resultados positivos. A quantidade média de carga bacteriana variou de 1,20 × 101 a 7,20 × 104, com média de 1,73 × 104 cópias. Das amostras reagentes, 81,81% (36 amostras) foram obtidas de abate não inspecionado, enquanto 18,18% (8 amostras) foram obtidas em abatedouro. Na análise histopatológica, foram avaliadas 44 amostras positivas para qPCR, das quais 28 (63,63%) apresentaram resultados compatíveis com o principal processo inflamatório associado à presença de M. hyopneumoniae, ou seja, hiperplasia do tecido linfóide associado ao brônquio (BALT). Três amostras que apresentaram maior carga bacteriana (qPCR: 5,63 × 10³, 2,19 × 104 e 7,23 × 104) foram mais evidentes neste estudo. Os achados microscópicos associados às quantificações indicaram uma relação entre a quantidade de carga bacteriana e a presença de lesão microscópica; a maior carga bacteriana no tecido pulmonar está associada a maior alteração histopatológica para hiperplasia BALT. Em conclusão, os resultados obtidos sinalizam para a circulação do agente etiológico nos animais amostrados e a necessidade de medidas preventivas nas criações de suínos do estado do Maranhão com envolvimento dos produtores, órgãos de defesa sanitária e inspeção.

Molecular prevalence of Mycoplasma parvum in production cycle of technified swine herds / Prevalência molecular do Mycoplasma parvum em granjas de suínos tecnificados de ciclo completo

Porcine hemoplasmosis is characterized as a geographically cosmopolitan disease caused by Mycoplasma suis and Mycoplasma parvum. Asymptomatic pigs are considered the focus of hemoplasmosis because they are carriers and reservoirs to new infections. This study aimed to determine the molecular occurrence of porcine hemoplasmas (PH) in the production cycle of technified farrow-to-finished swine herds. For this purpose, 20 swine herds were evaluated, where 501 whole blood samples were collected for qPCR and phylogenetic analyses for hemoplasmas. The epidemiological analysis was performed for the entire population and per the growth stage. The total prevalence for PH was 31.93% (161/501); 95% (19/20) of sampled herds were positive. The occurrence of PH by swine growth stages was nursery (30.47%), growing (31.29%), finishing (26.18%), and slaughter (40.25%). The quantification cycles (Cq) ranged from 3.18-39.56 and the number of PH 16S rRNA copies per µL of DNA ranged from 5,57 x10 to 2.23 x1010 . Sequencing and phylogenetic analysis of five selected samples showed 100% identity with M. parvum strain Indiana and two M. parvum sequences from Brazil/Goiás. This is the first report on PH in technified herds in Southeastern Brazil by growth stages.

A hemoplasmose suína é uma doença geograficamente cosmopolita, causada por Mycoplasma suis e Mycoplasma parvum. Suínos assintomáticos são considerados foco de hemoplasmose por serem portadores e reservatórios de novas infecções. Este estudo teve como objetivo determinar a ocorrência molecular de hemoplasmas suínos (HP) no ciclo de produção de rebanhos suínos tecnificados. Foram avaliados 20 rebanhos suínos e coletadas 501 amostras de sangue total para qPCR e análises filogenéticas para hemoplasmas. A análise epidemiológica foi realizada pela população e por estágio de crescimento. A prevalência total de HP foi de 31,93% (161/501); 95% (19/20) dos rebanhos amostrados foram positivos. A ocorrência de HP por fases de crescimento dos suínos foi: creche (30,47%), em crescimento (31,29%), acabamento (26,18%) e abate (40,25%). Os ciclos de quantificação (Cq) variaram de 3,18-39,56 e o número de cópias de rRNA PH 16S por µL de DNA variou de 5,57 x102 a 2,23 x10¹0. O sequenciamento e a análise filogenética de cinco amostras selecionadas mostraram 100% de identidade com a cepa indiana de M. parvum e duas sequências de M. parvum do Brasil / Goiás. Este é o primeiro relato de HP em rebanhos tecnificados, na região Sudeste do Brasil, por estágios de crescimento.

Canine ringworm caused by Trichophyton mentagrophytes - detection by SYBR-Green real-time PCR

Background: Dermatophytes, fungi of universal distribution, invade semi or fully keratinized structures, such as skin, fur/ hair and nails. The various species of dermatophytes are classified into three genera anamorphic: Microsporum, Trichophyton and Epidermophyton. The genus Epidermophyton includes only E. floccosum, that rarely affects animals. The main species responsible for the disease in dogs and cats are Microsporum canis, M. gypseum and Trichophyton mentagrophytes, which were characterized through conventional mycological methodology (microscopic examination with KOH and culture). Molecular methodologies, such as real-time PCR, can contribute to a rapid laboratory diagnosis, helping clinicians to initiate an early antifungal treatment. This case report describes a case of canine dermatophytosis due to Trichophyton mentagrophytes detected from a clinical sample by SYBR-Green real-time PCR. Case: A 8-year-old dog, rescued from the street, was referred to a private veterinary clinic in the city of Canoas, RS, Brazil, presenting generalized lymphadenomegaly, crusted lesions all over the body, generalized alopecia, signs of excoriation and epistaxis. Initially, were administered prednisone [1 mg/kg every 48 h, BID] and cephalexin [30 mg/kg, BID]. Weekly baths with benzoyl peroxide were also given. The therapy was not clinically successful. Wood's Lamp Test was negative. As a differential diagnosis, PCR for detection of Leishmania was negative. Complete blood count and serum biochemical assay were also performed. For mycological diagnosis, hair specimen was clarified and examined microscopically using 10% potassium hydroxide (KOH) for the visualization of chains of arthroconidia (ectothrix invasion of hair). The infected hair was plated onto MycoselTM Agar, incubated at 28°C for 15 days. Microscopy of hyphae/ conidia and macroscopic colony characteristics (colors and texture) were conducted for the differentiation of the species within the genus Microsporum and Trichophyton. In addition, real-time PCR was applied for direct analysis of the fungal DNA obtained from the hair sample. Microscopic examination was negative. The dermatophyte present in the hair sample was confirmed as Trichophyton mentagrophytes by culture and qPCR (melting-point analysis). The patient was treated with systemic itraconazole [10 mg/ kg SID - 90 days]. Twice-weekly application of 2.5 % miconazole and 2% chlorhexidine shampoo until complete cure. Discussion: Dermatophytosis is often listed as self-limiting infection; however, animal dermatophytosis can spread between pets, as well as a zoonotic transmission to humans. The literature on dermatophytosis indicates that Microsporum canis is the predominant etiological agent, followed by M. gypseum. Trichophyon mentagrophytes that appear in a lower percentage of isolation. The culture of hair, even with specific medium containing chloramphenicol and cyclohexamide, may present contaminating fungi, not related to dermatophytosis, which can inhibit or override the growth of dermatophytes. The use of real-time PCR provided a faster and specific diagnosis of dermatophytosis when compared to the conventional mycological methodology for detection and identification of T. mentagrophytes, which takes around 10 to 15 days for culture. It is possible to use this technique as an alternative diagnosis for dermatophytes associated to clinical hair samples of dogs.

PCV3-associated reproductive failure in pig herds in Brazil.

Porcine circovirus type 3 (PCV3) has been widely detected worldwide in healthy and sick pigs. Recently its association with clinical disease and reproductive failure has been proven through the detection of intralesional viral mRNA in affected pigs. This study aims to describe the occurrence of PCV3-associated reproductive failure (abortions) in sow herds in southern Brazil. Eleven fetuses from five different litters from two herds were analyzed. These herds reported an increase in the rate of late-gestation abortions, stillbirths, and the percentage of mummified piglets. At gross examination, six of the fetuses had large caudally rotated ears and one fetus was mummified. Microscopically, multisystemic vasculitis, lymphocytic interstitial pneumonia, myocarditis, and encephalitis were observed. These six fetuses with gross and histological lesions were positive in qPCR analysis for PCV3, and PCV3 transcription was shown through in situ hybridization (ISH-RNA) within the histologic lesions. Samples from all 11 fetuses tested negative in PCR exam for Porcine Circovirus type 1 and 2, Porcine Reproductive and Respiratory Syndrome, Porcine Parvovirus, and Atypical Porcine Pestivirus. Furthermore, based on the ORF2 analysis, the PCV3a clade was identified. This is the first report of PCV3a-associated reproductive failure in pig herds in South America.

Development of a droplet digital PCR assay for quantification of the proviral load of bovine leukemia virus.

Droplet digital PCR (ddPCR) is a highly sensitive tool developed for the detection and quantification of short-sequence variants-a tool that offers unparalleled precision enabling measurement of smaller-fold changes. We describe here the use of ddPCR for the detection of Bovine leukemia virus (BLV) DNA provirus. Serum samples and whole blood from experimentally infected sheep and naturally infected cattle were analyzed through ddPCR to detect the BLV gp51 gene, and then compared with serologic and molecular tests. The ddPCR assay was significantly more accurate and sensitive than AGID, ELISA, nested PCR, and quantitative PCR. The limit of detection of ddPCR was 3.3 copies/µL, detecting positive experimentally infected sheep beginning at 6 d post-infection. The ddPCR methodology offers a promising tool for evaluating the BLV proviral load, particularly for the detection of low viral loads.

Novel duplex RT-qPCR for animal rabies surveillance.

Rabies is a lethal zoonosis affecting mammals worldwide. Diagnosis of rabies follows international standard protocols, primarily relying on direct immunofluorescence (DI) followed by mouse inoculation test (MIT). WHO recommends molecular biology techniques such as RT-qPCR for replacing MIT to diagnose rabies in animal samples. Recently, a real-time PCR protocol that detects all rabies virus variants identified worldwide was validated. This assay is a pan-Lyssavirus TaqMan quantitative RT-PCR called LN34. A modified LN34 assay protocol was tested at the Paraná State Reference Laboratory (Lacen/PR) using animal samples previously tested by DI and MIT, the gold standard (GS). This method has been changed to a RT-qPCR duplex format to better fit the diagnostic routine. The new assay was called duplex LN34 and ß-actin RT-qPCR. All the 88 samples evaluated using the GS test, modified pan-Lyssavirus TaqMan RT-qPCR and duplex LN34 and ß-actin RT-qPCR showed 100% agreement with each other. This novel duplex RT-qPCR protocol has shown adequate diagnostic performance and may be used in research and surveillance purposes, replacing the standard MIT and ending mice use for rabies diagnosis.

Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples.

The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples.

Molecular investigation of haemotropic mycoplasmas and Coxiella burnetii in free-living Xenarthra mammals from Brazil, with evidence of new haemoplasma species.

Although mammals of the superorder Xenarthra are considered hosts of a wide range of zoonotic agents, works aiming at investigating the role of these animals as hosts for bacteria with zoonotic potential are rare. The present study aimed to investigate the occurrence and molecularly characterize Coxiella burnetii and haemoplasma (haemotropic mycoplasmas) DNA in blood and spleen samples from 397 free-living Xenarthra mammals (233 sloths, 107 anteaters and 57 armadillos) in five Brazilian states (Mato Grosso do Sul, São Paulo, Pará, Rondônia and Rio Grande do Sul). All biological samples from Xenarthra were negative in the qPCR for Coxiella burnetii based on the IS1111 gene. The absence of C. burnetii DNA in blood and spleen samples from Xenarthra suggests that these mammals may not act as possible hosts for this agent in the locations studied. When performed conventional PCR assays for the endogenous (gapdh) mammalian gene, 386 samples were positive. When screened by molecular assays based on the 16S rRNA gene of haemoplasmas, 81 samples were positive, of which 15.54% (60/386) were positive by conventional PCR and 5.44% (21/386) were positive by real-time PCR; three samples were positive in both assays. Of these, 39.74% (31/78) were also positive for the 23S rRNA gene and 7.69% (6/78) for the haemoplasma RNAse P gene. Among the samples positive for haemoplasmas, 25.64% (20/78) were obtained from anteaters (Tamandua tetradactyla and Myrmecophaga tridactyla), 39.74% (31/78) from sloths (Bradypus tridactylus, Bradypus sp. and Choloepus sp.) 34.61% (27/78) from armadillos (Priodontes maximus, Euphractus sexcinctus and Dasypus novemcinctus). A haemoplasma 16S rRNA sequence closely related and showing high identity (99.7%) to Mycoplasma wenyonii was detected, for the first time, in B. tridactylus. Based on the low identity and phylogenetic positioning of 16S rRNA and 23S rRNA sequences of haemoplasmas detected in anteaters and armadillos, the present study showed, for the first time, the occurrence of putative new Candidatus haemotropic Mycoplasma spp. ("Candidatus Mycoplasma haematotetradactyla" and "Candidatus Mycoplasma haematomaximus") in Xenarthra mammals from Brazil.

International interlaboratory validation of a nested PCR for molecular detection of Babesia bovis and Babesia bigemina, causative agents of bovine babesiosis.

Babesia bovis and B. bigemina are tick-transmitted parasites causing bovine babesiosis, characterized by significant morbidity and mortality leading to economic losses to the livestock industry in tropical and subtropical regions worldwide. Animals that recover from acute infection remain carriers with low parasitemia acting as a source of transmission, and often escape detection. An improved diagnosis of a B. bovis and/or B. bigemina infection of carrier animals is enabled by the availability of detection methods with high sensitivity. To this end, two nested PCR assays targeting the cytochrome b (cytb) genes of B. bovis and B. bigemina (cytb-nPCR), have been recently developed and an increased sensitivity with respect to reference protocols has been shown (Romero-Salas et al., 2016). In this study, the specificity against a panel of hemoparasites that potentially co-occur with B. bovis and B. bigemina was demonstrated to ensure applicability of the cytb-nPCR assays in a wide range of regions where bovine babesiosis is endemic. Furthermore, we compared both reported cytb-nPCR assays with reference nPCR and qPCR protocols for (i) their capability to detect carrier animals in the field, and (ii) their reproducibility when performed in different laboratories by independent operators. We show that, in a panel of bovine field samples (n = 100), the cytb-nPCR assays detected a considerably higher number of 25% B. bovis and 61% B. bigemina-positive animals compared to 7% and 20% B. bovis and 55% and 49% B. bigemina-positive animals when tested by reference nPCR and qPCR protocols, respectively. Cytb-nPCRs were also found superior in the detection of carrier animals when field samples from Africa were analyzed. In addition, both the B. bovis and B. bigemina cytb-nPCR assays were independently validated in a single blinded study in three laboratories. Importantly, no significant differences in the number/percentage of infected animals was observed using cytb-nPCR assays. In summary, the cytb-nPCR assays detected a considerably higher number of chronically infected B. bovis and B. bigemina carrier animals compared to reference nPCR and qPCR protocols, when applied in different epidemiological field situations. Furthermore, a high reproducibility between laboratories could be demonstrated.