First report of Giardia duodenalis in pet rabbits in Brazil.

Giardia duodenalis is a flagellate protozoan that multiplies in the small intestine of a wide variety of hosts, animals and humans. It has a worldwide distribution, however it is considered a neglected disease by the World Health Organization (WHO). Nowadays, rabbits are being chosen as pets, especially by children. There are already reports of the occurrence of G. duodenalis in rabbits from other countries, but research has not been carried out in Brazil yet. Thus, the objective of our work was to verify the occurrence and molecularly characterize G. duodenalis that affect pet rabbits, through the polymerase chain reaction (PCR) in the northwest region of the state of São Paulo, Brazil. Fecal samples from 100 rabbits were collected, which later underwent a process of DNA extraction and amplification by nested-PCR (nPCR), using the SSU rRNA gene, and ß-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) to determine the assemblage. A questionnaire was answered by the owners with information about gender, age, deworming, diarrhea, water source, food, place of residence and contact with other animals. From those samples, 40 were positive for G. duodenalis. Good quality of the SSU rRNA gene by nPCR were obtained from two samples. For the first time, we report the occurrence of G. duodenalis assemblage A on pet rabbits in Brazil.

Toward automating the diagnosis of gastrointestinal parasites in cats and dogs.

Diagnosing gastrointestinal parasites by microscopy slide examination often leads to human interpretation errors, which may occur due to fatigue, lack of training and infrastructure, presence of artifacts (e.g., various types of cells, algae, yeasts), and other reasons. We have investigated the stages in automating the process to cope with the interpretation errors. This work presents advances in two stages focused on gastrointestinal parasites of cats and dogs: a new parasitological processing technique, named TF-Test VetPet, and a microscopy image analysis pipeline based on deep learning methods. TF-Test VetPet improves image quality by reducing cluttering (i.e., eliminating artifacts), which favors automated image analysis. The proposed pipeline can identify three species of parasites in cats and five in dogs, distinguishing them from fecal impurities with an average accuracy of 98,6%. We also make available the two datasets with images of parasites of dogs and cats, which were obtained by processing fecal smears with temporary staining using TF-Test VetPet.

TF-Test techniques for the laboratory diagnosis of gastrointestinal parasites of humans and animals

Intestinal parasites inhabit the intestinal tract of humans and animals, causing damages whose severity depends on several factors related to the parasite and the host. Immunocompromised individuals are more likely to develop severe forms of parasitic infestation. The diagnosis of the gastrointestinal parasitosis is mainly performed by the examination of the feces, which consists of the direct visualization and identification of the parasites eliminated through the feces. These tests are generally low sensitive and the microscope slides contain a large number of impurities, which can impair the result of the diagnosis. In order to improve the diagnostic accuracy, a new parasitological technique called Three Fecal Test (TF­Test) was developed. To further improve its diagnostic accuracy, few modifications of the original protocols have been made with the years. In this study the performance of these new techniques to detect gastrointestinal parasites in human and animal fecal samples was described and discussed in relation to the performance of other conventional coprological tests. It could be concluded that the TF­Test conventional and modified can be used for the diagnosis of several human and animal parasites, with satisfactory results.

Optimizing the quality of research in Veterinary Parasitology.

Countless research is carried out until new discoveries are transformed into products or services available to the population. This trajectory can be slower and more costly or even impossible when irreproducible data are obtained in the most diverse fields of science. Thus, quality management appears as an essential tool to guarantee the reliability of academic research results. In this work, we demonstrate the applied strategy to implement a Quality Management System (QMS) in a research laboratory in Veterinary Parasitology and we highlight the adaptable quality requirements in this scientific research environment. For this, the Plan-Do-Check-Act (PDCA) quality tool was used, and two internal audits were performed, one before and one after implementation. The audits reached 67 (41.36%) and 157 (96.91%) points before and after implementation, respectively, with a significant difference between the moments studied. Thus, we demonstrate that the adoption of QMS principles in research is feasible. The methodology applied in this work can be adopted by managers from other laboratories interested in the implementation of quality standards as a support in the reproducibility of research.

Automated Diagnostics: Advances in the Diagnosis of Intestinal Parasitic Infections in Humans and Animals.

The increasingly close proximity between people and animals is of great concern for public health, given the risk of exposure to infectious diseases transmitted through animals, which are carriers of more than 60 zoonotic agents. These diseases, which are included in the list of Neglected Tropical Diseases, cause losses in countries with tropical and subtropical climates, and in regions with temperate climates. Indeed, they affect more than a billion people around the world, a large proportion of which are infected by one or more parasitic helminths, causing annual losses of billions of dollars. Several studies are being conducted in search for differentiated, more sensitive diagnostics with fewer errors. These studies, which involve the automated examination of intestinal parasites, still face challenges that must be overcome in order to ensure the proper identification of parasites. This includes a protocol that allows for elimination of most of the debris in samples, satisfactory staining of parasite structures, and a robust image database. Our objective here is therefore to offer a critical description of the techniques currently in use for the automated diagnosis of intestinal parasites in fecal samples, as well as advances in these techniques.

Cryptosporidiosis and Giardiasis in Buffaloes (Bubalus bubalis).

Cryptosporidium spp. and Giardia duodenalis infect the gastrointestinal tracts of animals and humans. Both parasite groups are distributed worldwide and cause significant economic losses in animal productivity. Infected hosts presenting with and without clinical manifestations can eliminate infective forms of these protozoa, which are particularly important to One Health. Compared to the published research on cattle, relatively few studies have examined the epidemiology of cryptosporidiosis and giardiasis in buffaloes. This short review describes the global occurrence of Cryptosporidium spp. and G. duodenalis in buffaloes, including the molecular techniques employed for the identification of species/assemblages and genotypes of these protozoa. Genetic analyses of isolates of G. duodenalis and Cryptosporidium spp. from various sources (environmental, animal, and human) have been performed to investigate their epidemiology. In buffaloes, the species Cryptosporidium parvum, Cryptosporidium ryanae, Cryptosporidium bovis, and Cryptosporidium suis-like have been characterized, as well as assemblages A and E of G. duodenalis. We demonstrate that buffaloes can be infected by species of Cryptosporidium spp. and G. duodenalis assemblages with zoonotic potential. Epidemiological studies that utilize molecular biology techniques represent an important resource for efforts to control and prevent the spread of these protozoans.

A historical review of the techniques of recovery of parasites for their detection in human stools.

Since the early 20th century, the detection of intestinal parasites has improved with the development of several techniques for parasitic structures recovery and identification, which differ in sensitivity, specificity, practicality, cost, and infrastructure demand. This study aims to review, in chronological order, the stool examination techniques and discuss their advantages, limitations, and perspectives, and to provide professionals and specialists in this field with data that lays a foundation for critical analysis on the use of such procedures. The concentration procedures that constitute the main techniques applied in routine research and in parasitological kits are a) spontaneous sedimentation; b) centrifugation-sedimentation with formalin-ethyl acetate; and c) flotation with zinc sulfate solution. While selecting a technique, one should consider the purpose of its application and the technical-operational, biological, and physicochemical factors inherent in the procedures used in stool processing, which may restrict its use. These intrinsic limitations may have undergone procedural changes driven by scientific and technological development and by development of alternative methods, which now contribute to the improvement of diagnostic accuracy.

Automated Diagnosis of Canine Gastrointestinal Parasites Using Image Analysis.

Because canine intestinal parasites are considered cosmopolitan, they carry significant zoonotic potential to public health. These etiological agents are routinely diagnosed using microscopic examination commonly used because of its low cost, simple execution, and direct evidence. However, there are reports in the literature on the poor performance of this test due to low to moderate sensitivity resulting from frequent errors, procedures and interpretation. Therefore, to improve the diagnostic efficiency of microscopic examination in veterinary medicine, we developed and evaluated a unique new protocol. This system was tested in a study involving four genera of highly prevalent canine intestinal parasites in an endemic region in São Paulo state, Brazil. Fecal samples from 104 animals were collected for this research. The new protocol had a significantly higher (p < 0.0001) number of positive cases on image data, including parasites and impurities, and was elaborate to test them with the TF-GII/Dog technique, with a moderate agreement and Kappa index of 0.7636. We concluded that the new Prototic Coproparasitological Test for Dogs (PC-Test Dog) allowed a better visualization of the parasitic structures and showed a favorable result for the diagnosis of intestinal parasites in dogs.

A historical review of the techniques of recovery of parasites for their detection in human stools

Abstract Since the early 20th century, the detection of intestinal parasites has improved with the development of several techniques for parasitic structures recovery and identification, which differ in sensitivity, specificity, practicality, cost, and infrastructure demand. This study aims to review, in chronological order, the stool examination techniques and discuss their advantages, limitations, and perspectives, and to provide professionals and specialists in this field with data that lays a foundation for critical analysis on the use of such procedures. The concentration procedures that constitute the main techniques applied in routine research and in parasitological kits are a) spontaneous sedimentation; b) centrifugation-sedimentation with formalin-ethyl acetate; and c) flotation with zinc sulfate solution. While selecting a technique, one should consider the purpose of its application and the technical-operational, biological, and physicochemical factors inherent in the procedures used in stool processing, which may restrict its use. These intrinsic limitations may have undergone procedural changes driven by scientific and technological development and by development of alternative methods, which now contribute to the improvement of diagnostic accuracy.

Use of the aqueous biphasic system as an alternative for concentration of Ascaris lumbricoides eggs, with non-toxic separation of faecal residues and fats.

OBJECTIVES: Human enteroparasites are considered a serious public health problem in underdeveloped countries located in world regions with tropical, subtropical and equatorial climates. These parasites are commonly diagnosed by the Parasitological Examination of Faeces (PEF), performed by conventional techniques and/or commercial kits that result in tests with low-to-moderate sensitivity, due to the use of destructive chemical solvents to parasite structures, and to present excess adipose substance and digestive residues in their microscopic slides. In order to improve the efficacy of these tests/examinations, this study aimed to investigate a new alternative for the PEF, with the use of Aqueous Biphasic System (ABS). METHODS: For this, four ABSs containing poly (ethylene glycol), PEG (PEG-4000 and PEG-6000), dipotassium phosphate and sodium citrate at different concentrations in the biphasic systems were evaluated with faecal samples containing eggs of Ascaris lumbricoides. RESULTS: The ABS consisting of PEG-4000 and dipotassium phosphate, at concentrations of 55% w/w and 20% w/w, respectively, achieved 100% satisfactory results compared to the conventional TF-Test technique in terms of preservation and concentration of A. lumbricoides eggs, with adequate separation of digestive residues, without using a centrifuge or chemical solvents that may cause harm to the parasites. CONCLUSIONS: This study presents ABS as a new low-cost technical principle for the detection of parasite eggs in PEF. The new technique is simple, fast, non-toxic, not harmful to the parasite and does not require a centrifuge.

UTILISATION DU SYSTÈME BIPHASIQUE AQUEUX COMME ALTERNATIVE À LA CONCENTRATION DES ŒUFS D'ASCARIS LUMBRICOIDES, AVEC SÉPARATION NON TOXIQUE DES RÉSIDUS FÉCAUX ET DES GRAISSES: OBJECTIFS: Les entéroparasites humains sont considérés comme un sérieux problème de santé publique dans les pays sous-développés situés dans des régions du monde aux climats tropicaux, subtropicaux et équatoriaux. L'examen parasitologique des matières fécales (EPMF) permet généralement de diagnostiquer ces parasites, à l'aide de techniques classiques et/ou de kits commerciaux, conduisant à des tests de sensibilité faible à modérée, dus à l'utilisation de solvants chimiques destructeurs pour les structures du parasite et résultant en excès de substances adipeuses et de résidus digestifs dans leurs lames microscopiques. Afin d'améliorer l'efficacité de ces tests/examens, cette étude visait à étudier une nouvelle alternative à l'EPMF, avec l'utilisation du Système Biphasique Aqueux (SBA). MÉTHODES: Pour cela, quatre SBA contenant du poly (éthylène glycol), du PEG (PEG-4000 et PEG-6000), du phosphate dipotassique et du citrate de sodium à différentes concentrations ont été évalués avec des échantillons de matières fécales contenant des œufs d'Ascaris lumbricoides. RÉSULTATS: Le SBA constitué de PEG-4000 et de phosphate dipotassique, à des concentrations respectives de 55% poids/poids et 20% poids/poids, a obtenu des résultats satisfaisants à 100% par rapport à la technique conventionnelle du TF-Test, en termes de préservation et de concentration des œufs de A. lumbricoides, avec une séparation adéquate des résidus digestifs, sans utiliser de centrifugeuse ni de solvants chimiques pouvant détruire les parasites. CONCLUSIONS: Cette étude présente le SBA comme un nouveau principe technique peu coûteux pour la détection des œufs de parasites dans l'EPMF. La nouvelle technique est simple, rapide, non toxique, n'endommage pas le parasite et ne nécessite pas de centrifugeuse.

Prevalence of Ehrlichia canis (Rickettsiales: Ehrlichieae) DNA in Tissues From Rhipicephalus sanguineus (Acari: Ixodidae) Ticks in Areas Endemic for Canine Monocytic Ehrlichiosis in Brazil.

Canine monocytic ehrlichiosis (CME) is a disease caused by the obligate intracellular bacterium Ehrlichia canis. Tropical lineages of Rhipicephalus sanguineus ticks play an essential role in the transmission of this pathogen. The aim of the present study was to evaluate the prevalence of E. canis DNA in tissue from R. sanguineus ticks in areas endemic for CME in Brazil and quantify levels of E. canis DNA in dissected tissues from these samples. A total of 720 ticks were collected from 72 dogs (36 dogs from the city Araçatuba in São Paulo state and 36 from Campo Grande in the state of Mato Grosso do Sul). Ticks were dissected to collect the guts, ovaries and salivary gland. A quantitative polymerase chain reaction (qPCR) targeting the disulphide bond formation (dsb) protein gene was performed to quantify the level of E. canis infection. The E. canis dsb-qPCR assay was positive for 31.9, 10, and 15.2% of the gut, ovary, and salivary glands, respectively. The average gut, ovary, and salivary gland bacterial load estimated by qPCR was 1.21 × 103, 2.60 × 103, and 4.92 × 103 gene copies/µl, respectively. This is the first report of E. canis DNA in ovaries of R. sanguineus ticks parasitizing dogs in these CME-endemic areas. These observations raise the possibility of E. canis trans-ovarial transmission.

First description of Giardia duodenalis in buffalo calves (Bubalus bubalis) in southwest region of São Paulo State, Brazil.

We performed molecular characterization of Giardia duodenalis in buffalo calves from the Southwest region of São Paulo State, Brazil. A total of 183 fecal samples of Murrah breed buffaloes up to six months of age were collected. We examined these samples by the polymerase chain reaction (PCR) targeting the small-subunit ribosomal RNA gene and positive samples were characterized using additional PCR assays targeting a portion of the beta-giardin, the glutamate dehydrogenase and the triose-phosphate isomerase genes. Based on the SSU rRNA nPCR, the presence of G. duodenalis was confirmed in 12 (6.56%) of fecal samples, of these, five, four and three samples were positive for the tpi, bg and gdh genes, respectively. Assemblage identification by sequencing was successful in 6 of 12 samples and sequence analysis showed 100% genetic similarity with G. duodenalis assemblage E. This observation represents the first detection of G. duodenalis assemblage E in buffaloes calves in Brazil.

First description of Cryptosporidium hominis GP60 genotype IkA20G1 and Cryptosporidium parvum GP60 genotypes IIaA18G3R1 and IIaA15G2R1 in foals in Brazil.

The present study focuses on Cryptosporidium infections of foals in Brazil. A total of 92 animals of different breeds from 11 farms in the vicinity of Araçatuba in the state of São Paulo, were examined. According to PCR targeting the 18S rRNA gene, Cryptosporidium sp. DNA was detected in 21.7% (20/92) of foals. Good quality 18S rRNA, actin, HSP70 and gp60 genes nPCR amplicons were obtained from five fecal samples. PCR amplification and sequencing of a fragment of the GP60 sporozoite surface glycoprotein gene revealed C. parvum genotypes IIaA18G3R1, IIaA15G2R1. Interestingly, we also detected in two foals a GP60 genotype related to the human parasite C. hominis.

Validation of a new technique to detect Cryptosporidium spp. oocysts in bovine feces.

Due to its important zoonotic potential, cryptosporidiosis arouses strong interest in the scientific community, because, it was initially considered a rare and opportunistic disease. The parasitological diagnosis of the causative agent of this disease, the protozoan Cryptosporidium spp., requires the use of specific techniques of concentration and permanent staining, which are laborious and costly, and are difficult to use in routine laboratory tests. In view of the above, we conducted the feasibility, development, evaluation and intralaboratory validation of a new parasitological technique for analysis in optical microscopy of Cryptosporidium spp. oocysts, called TF-Test Coccidia, using fecal samples from calves from the city of Araçatuba, São Paulo. To confirm the aforementioned parasite and prove the diagnostic efficiency of the new technique, we used two established methodologies in the scientific literature: parasite concentration by centrifugal sedimentation and negative staining with malachite green (CSN-Malachite) and Nested-PCR. We observed good effectiveness of the TF-Test Coccidia technique, being statistically equivalent to CSN-Malachite. Thus, we verified the effectiveness of the TF-Test Coccidia parasitological technique for the detection of Cryptosporidium spp. oocysts and observed good concentration and morphology of the parasite, with a low amount of debris in the fecal smear.

Ocorrência da infecção por Cryptosporidium spp. em cabritos (Capra hircus) / Occurrence of infection by Cryptosporidium spp. in goat kids (Capra hircus)

O presente estudo teve como objetivo determinar a ocorrência da infecção por Cryptosporidium spp. em cabritos de Quixadá, Ceará, Brasil. Participaram do estudo 400 cabritos, com idade entre três e 360 dias, de ambos os sexos, com e sem padrão racial definido, procedentes de 25 estabelecimentos rurais distribuídos em três circuitos. As fezes foram cadastradas de acordo com o aspecto e cor, distribuídas em tubos tipo "eppendorf®" e congeladas in natura a -20°C, até o momento das extrações de DNA genômico do parasito com auxílio de kit comercial. Para amplificação de fragmentos da subunidade 18S do RNA ribossômico (rRNA) foi utilizada a "Nested"-PCR. A ocorrência de Cryptosporidium spp em cabritos de Quixadá foi de 7,50% (30/400). A frequência no período seco e no chuvoso foi de 9,55% (19/199) e 5,47% (11/201), respectivamente (χ²=2,39 e P>0,05). Amostras positivas foram identificadas em 64,00% (16/25) das propriedades estudadas e dessas amostras 50,00% (15/30) e 70,00% (21/30) tinham as fezes com aspecto e cor normais, respectivamente, sugerindo que cabritos assintomáticos estão eliminando oocistos. Não foi observada positividade para Cryptosporidium spp. em animais com 301 a 360 dias, demonstrando que animais mais velhos apresentam menos possibilidade de se infectarem com o parasito...

The present study aimed to determine the occurrence of infection by Cryptosporidium spp. in goat kids from Quixadá, Ceará, Brazil. The study included 400 goat kids of both sexes, 3 to 360 days old, with or without defined breed, originating from 25 farms distributed in three circuits. Feces were registered in accordance with the appearance and color, distributed into tubes Eppendorf tubes and frozen in natura at-20°C until the moment of extraction of genomic DNA from the parasite with the aid of a commercial kit. For amplification of fragments of the 18S subunit of ribosomal RNA (rRNA) was used to Nested PCR. The occurrence of Cryptosporidium spp. in goats kids of the Quixadá was 7.50% (30/400). The frequency in the dry period and rainy was 9.55% (19/199) and 5.47% (11/201) respectively (χ²=2.39 and P>0.05). Positive samples were identified in 64.00% (16/25) of the studied farms, and from these samples 50.00% (15/30) and 70.00% (21/30) had feces with normal appearance and color respectively, suggesting that the asymptomatic goats were eliminating oocysts. No positivity for Cryptosporidium spp. was observed in 301 to 360-day-old goats, demonstrating that older animals have less chance to become infected with the parasite...

Cryptosporidium spp. infection in mares and foals of the northwest region of São Paulo State, Brazil / Infecção por Cryptosporidium spp. em éguas e potros da região noroeste do estado de São Paulo, Brasil

The present study aimed to analyze the occurrence of infection by Cryptosporidium spp. in mares and their respective foals. This study was carried out in 11 farms located in the municipalities of Araçatuba, Birigui, Guararapes and Santo Antônio do Aracangua, in the northwest region of the State of Sao Paulo, from November 2010 to March 2011. A total of 98 mares and 98 foals of several breeds were analyzed; among foals, 59 were males and 39 females, aged from three to 330 days. Feces were collected directly from the rectal ampulla, purified and processed according to modified Kinyoun stain. Occurrence of Cryptosporidium spp. was 21.4% (21/98) for foals and 18.4% (18/98) for mares. Occurrence of Cryptosporidium spp. had significant association with breeds and age of animals. Results obtained led to the conclusion that foals older than two months and Mangalarga animals are less susceptible to the occurrence of Cryptosporidium spp(AU)

O presente estudo teve como objetivo analisar a ocorrência da infecção por Cryptosporidium spp. em éguas e seus respectivos potros. Este estudo foi realizado em 11 fazendas localizadas nos municípios de Araçatuba, Birigui, Guararapes e Santo Antônio do Aracangua, na região Noroeste do Estado de São Paulo, de novembro de 2010 a março de 2011. Um total de 98 éguas e 98 potros de diversas raças foram analisados, sendo que, entre os filhotes, 59 eram machos e 39 fêmeas, cujas idades variavam de três até 330 dias. Fezes foram colhidas diretamente da ampola retal, purificadas e processadas pela técnica de Kinyoun modificada. A ocorrência de Cryptosporidium spp. observada foi de 21,4% (21/98) para potros e 18,4% (18/98) para éguas. A ocorrência de Cryptosporidium spp. teve uma associação significativa com a raça e a idade dos animais. A partir dos resultados obtidos, conclui-se neste estudo que potros com idade superior a dois meses e animais da raça Mangalarga foram menos susceptíveis à ocorrência de Cryptosporidium spp(AU)

Cryptosporidium spp. infection in mares and foals of the northwest region of São Paulo State, Brazil.

The present study aimed to analyze the occurrence of infection by Cryptosporidium spp. in mares and their respective foals. This study was carried out in 11 farms located in the municipalities of Araçatuba, Birigui, Guararapes and Santo Antônio do Aracangua, in the northwest region of the State of Sao Paulo, from November 2010 to March 2011. A total of 98 mares and 98 foals of several breeds were analyzed; among foals, 59 were males and 39 females, aged from three to 330 days. Feces were collected directly from the rectal ampulla, purified and processed according to modified Kinyoun stain. Occurrence of Cryptosporidium spp. was 21.4% (21/98) for foals and 18.4% (18/98) for mares. Occurrence of Cryptosporidium spp. had significant association with breeds and age of animals. Results obtained led to the conclusion that foals older than two months and Mangalarga animals are less susceptible to the occurrence of Cryptosporidium spp.