The Fasciola hepatica egg development and hatching test (EDHT) using commercial drugs: a simple protocol for tackling anthelminthic resistance and promoting cattle productivity.

Fasciolosis is an important zoonotic disease caused by the trematode Fasciola hepatica, and it causes great losses in bovine production. The anthelmintic resistance is a major problem in the control of fasciolosis. In this study, the F. hepatica egg development and hatching test (EDHT) was used for the evaluation of the ovicidal activity of commercial drugs, commonly used for treating infected cattle, which reflects F. hepatica anthelminthic resistance in infected bovines, according to recent literature. Bile samples from F. hepatica naturally parasitized cattle were obtained from slaughterhouses in the cities of Lages and Otacílio Costa, Santa Catarina State, Brazil. The bile was washed, the eggs were recovered, quantified, and distributed in universal collectors, with a minimum of 1,000 eggs per vial. Four commercial drugs were used in this study, containing albendazole sulfoxide (ABDZ), closantel (CSTL), nitroxynil (NTXL), and triclabendazole with fenbendazole (TBZF). The drugs were diluted according to the manufacturer instructions. All drugs, and the respective control, were tested in triplicates, with the quantity of recovered eggs determining the number of drugs to be tested. The vials were incubated for 28 days at 27 °C, and the eggs were classified according to their degree of development under a stereomicroscope. In total, 121 egg samples were analyzed. Two samples were identified as resistant to TBZF. Undetermined resistance/susceptibility has been found in two isolates treated with ABDZ, one treated with NTXL and six treated with TBZF. CSTL did not present ovicidal activity and cannot be used in EDHT. This is the first time that commercial drugs were used in F. hepatica EDHT.

New records of Amblyomma ticks parasitizing neotropical primates in Brazil.

Amblyomma is an important tick genus for animal and human health, with some species being the vectors of zoonotic pathogens, such as Rickettsia rickettsii, in the Neotropical region. Knowing their hosts may help to understand the distribution of these agents and decrease the occurrence of clinical cases. Primates are intelligent and adaptable animals that can get close to humans in the search for food. So, they may be an important epidemiological link for the spread of these ticks. Beyond that, primates also suffer from these infections, serving as sentinels for different diseases. Thus, the present study aims to report the parasitism by Amblyomma spp. on six species of Neotropical primates from different locations in Brazil. The 337 collected ticks were morphologically identified using stereomicroscopes and taxonomic keys, and six distinct species of ticks were identified. We report here the first record of nymphs of the tick species Amblyomma cajennense sensu stricto on Alouatta belzebul, a male of Amblyomma fuscum on Alouatta guariba clamitans, nymphs of Amblyomma sculptum on Leontopithecus chrysopygus and Callithrix aurita, as well as nymphs of Amblyomma geayi on Saimiri collinsi. Of the 337 tick specimens collected, 256 (75,96%) were nymphs. The importance of primates in the life cycle of these species remains to be elucidated.

Experimental infection with Neospora caninum in Texel ewes at different stages of gestation.

In this study Texel sheep, at different stages of pregnancy, were experimentally infected with Neospora caninum. Eleven ewes, seronegative for N. caninum and Toxoplasma gondii, were inoculated 30 days before breeding (Group A), or at 65, 100, and 120 days of gestation (Groups B, C, and D). The group E (control) was inoculated with PBS. Blood samples were collected at -2, 2, 5, and 7 days post-infection (dpi), and weekly up to 42 dpi, for hematology, parasitemia (PCR), and serology (RIFI) assessments. Blood and tissue samples were collected from the lambs for molecular and histological analyses. All animals in Groups B, C, and D were seroconverted, whilst those in groups A and E remained seronegative. Parasitic DNA was detected in the blood of two ewes (groups B and D) and a lamb (group D), and in the brain of a lamb (group B). The parasitemia-positive ewe in group B delivered a weak and seropositive lamb, and had parasitic DNA in its placenta. These results confirm the vertical transmission of N. caninum in ewes inoculated at the beginning and end of pregnancy. The absence of abortions and other clinical signs suggest that Texel sheep may potentially have resistance to N. caninum.

First report of canine rangeliosis in domestic dogs from different regions of Santa Catarina State, Brazil.

Rangeliosis is the disease caused by Rangelia vitalii, a parasite reported in dogs from southeastern and southern Brazil, Uruguay, Paraguay, and Argentina. This protozoan is transmitted by the ixodid Amblyomma aureolatum, and infects erythrocytes, leukocytes, and vascular endothelial cells of the host. The common clinical signs, such as prostration, fever, anemia, thrombocytopenia, anorexia, weight loss, and dehydration, are also found in other infections, like canine babesiosis and ehrlichiosis. The similar clinical presentation with other diseases, as well as the indistinguishable morphology with intraerythrocytic Babesia canis, complicates the disease diagnostic. In the present study, blood samples from dogs presenting clinical signs compatible with hemoparasitosis were investigated for rangeliosis. The dogs were treated at veterinary clinics in the cities of Blumenau and Lages, in the State of Santa Catarina, Brazil. Blood samples from 17 dogs were analyzed by PCR. The samples were screened by a conventional piroplasma-PCR and the positives confirmed by a specific R. vitalii-qPCR. Two animals (2/17; 11.8%) were positive for R. vitalii, one from Blumenau and the other from Lages. Both animals presented unspecific signs of hemoparasitosis, such as apathy, anemia, and anorexia. The results indicate the necessity of molecular assays for the proper identification of the hemoparasite, and to investigate the real prevalence of rangeliosis in the State of Santa Catarina.

Epidemiological Survey and Confirmation of Autochthonous Cases of Bovine Fasciolosis in the Serrana Mesoregion of Santa Catarina, Brazil.

Fasciola hepatica is reported as a trematode of great importance, causing fasciolosis, one of the most neglected zoonotic parasitic diseases in the world. In Brazil, potential losses of around US$ 210 million per year were calculated, with bovine fasciolosis alone. The State of Santa Catarina is located in the South Region of Brazil, with a herd of more than 4 million ruminants. The Serrana Mesoregion, historically free of fasciolosis, is composed of 30 municipalities, which have a consolidated agricultural tradition, mainly in extensive ruminant livestock. The objective of the present work was to investigate the occurrence and distribution of autochthonous cases of bovine fasciolosis in the region. For this, different analyzes were carried out in rural properties from eight municipalities, and in three slaughterhouses under the State Inspection Service (SIE), which received animals from all the Serrana Mesoregion. The rural properties were randomly selected, and up to 20 fecal samples were collected from random cattle older than 1 year, for analysis by coproparasitological sedimentation test. The traceability records of the positives were checked, for identifying autochthonous cases. Additionally, the tracking data of cattle slaughtered between the years of 2018 and 2021, which presented liver condemnations, were analyzed for defining autochthonous cases of bovine fasciolosis. In total, there were fecal samplings in 106 farms, with 1,927 tested cattle. From these, 178 (9.2%) were positive and autochthonous, but the overall result did not reflect the heterogeneity found between the evaluated municipalities. During the analyzed period, 11,556 cattle were slaughtered, with 1,744 (15.1%) liver condemnations due to fasciolosis in animals that never left the Serrana Mesoregion. The present study confirmed the presence of autochthonous cases of bovine fasciolosis in municipalities of the Serrana Mesoregion, and verified a high rate of positivity in animals slaughtered in the regional slaughterhouses. When the prevalence of bovine fasciolosis in vivo was compared with the postmortem positivity index in five municipalities, the results disagreed in four municipalities (p-value < 0.05), emphasizing the need for field investigation for the better understanding of the distribution and frequency of the parasitosis.

Prevalence and risk factors for Dirofilaria immitis in dogs from Laguna, Santa Catarina, Brazil.

Dirofilaria immitis is a nematode found worldwide and able to infect several species, such as dogs, cats and, under rare circumstances, humans. In Brazil, most of the infections are related to coastal areas. The present study aimed at determining Dirofilaria immitis prevalence and evaluating the possible risk factors for infection in dogs, in Laguna, Santa Catarina, Brazil. Blood samples from 238 dogs were collected and a questionnaire was applied to the owners in order to obtain general information about the animals (age, gender, breed, coat color, home location (region), the use of dewormer/ectoparasiticide drugs, habits and behavior, roaming, insect control and the owner's knowledge about the disease). Samples were processed by blood smear techniques, modified Knott's test, immunochromatographic assay and PCR. Data were statistically analyzed by χ2 and OR. 4.62% was the overall prevalence of canine dirofilariasis and the suburb of Bananal, in Laguna, showed the highest prevalence (18.8%) when comparing with other regions (p < 0.05). However, there was no other significant factor related to the prevalence of D. immitis infection. Thus, the research have confirmed the presence of D. immitis in Laguna region, with a high prevalence in Bananal suburb, a dense forested area.

Secondary Metabolites of Pseudomonas aeruginosa LV Strain Decrease Asian Soybean Rust Severity in Experimentally Infected Plants.

Asian Soybean Rust (ASR), a disease caused by Phakopsora pachyrhizi, causing yield losses up to 90%. The control is based on the fungicides which may generate resistant fungi. The activation of the plant defense system, should help on ASR control. In this study, secondary metabolites of Pseudomonas aeruginosa LV strain were applied on spore germination and the expression of defense genes in infected soybean plants. The F4A fraction and the pure metabolites were used. In vitro, 10 µg mL-1 of F4A reduced spore germination by 54%, while 100 µg mL-1 completely inhibited. Overexpression of phenylalanine ammonia lyase (PAL), O-methyltransferase (OMT) and pathogenesis related protein-2 (PR-2; glucanases) defense-related genes were detected 24 and 72 h after soybean sprouts were sprayed with an organocopper antimicrobial compound (OAC). Under greenhouse conditions, the best control was observed in plants treated with 60 µg mL-1 of PCA, which reduced ASR severity and lesion frequency by 75% and 43%, respectively. Plants sprayed with 2 and 20 µg mL-1 of F4A also decreased severity (41%) and lesion frequency (32%). The significant reduction in spore germination ASR in plant suggested that the strain of these metabolites are effective against P. pachyrhizi, and they can be used for ASR control.

Parasites in road-killed wild felines from North of Paraná state, Brazil.

This study aimed to identify the intestinal parasites of road-killed wild felines in the North Central and North, Paraná state, southern Brazil. The animals were monitored by sampling previously established transects. The places where the felines were run over were mapped, the animals were identified, and the gastrointestinal tract was evaluated. The feces were submitted to coproparasitological techniques of spontaneous sedimentation, floating in hypersaturated NaCl solution and centrifugal floating in zinc sulfate. All the parasitic structures detected were photomicrographed. In the coproparasitological analyses were identified oocysts of Cystoisospora spp., eggs of Ancylostomatidae, and Capillaria spp.; eggs of Aelurostrongylus spp., Toxocara spp., Physaloptera spp., Taenia spp., and Spirometra spp.; Aelurostrongylus abstrusus larvae; and eggs and adults of Ancylostoma cati and Taenia spp. One of the cats was parasitized by a flea of Ctenocephalides felis felis. Based on these results, the animals analyzed in this study supplied important samples for the evaluation of parasitic diversity of North of Paraná and suggested that this region may have conditions that allow the maintenance of these parasites life cycles in the environment and among wildlife.

Corrigendum: Determining the Targets of Fluopsin C Action on Gram-Negative and Gram-Positive Bacteria.

[This corrects the article DOI: 10.3389/fmicb.2020.01076.].

Determining the Targets of Fluopsin C Action on Gram-Negative and Gram-Positive Bacteria.

The antibiotic activity of metalloantibiotic compounds has been evaluated since the 90s, and many different modes of action were characterized. In the last decade, the effects of secondary metabolites produced by Pseudomonas aeruginosa LV strain, including a cupric compound identified as Fluopsin C, were tested against many pathogenic bacteria strains, proving their high antibiotic activity. In the present study, the bactericidal mechanisms of action of Fluopsin C and the semi-purified fraction F4A were elucidated. The results found in electron microscopy [scanning electron microscopy (SEM) and transmission electronic microscopy (TEM)] demonstrated that both Fluopsin C and F4A are affecting the cytoplasmatic membrane of Gram-positive and Gram-negative bacteria. These results were confirmed by fluorescence microscopy, where these bacteria presented permeabilization of their cytoplasmatic membranes after contact with the semi-purified fraction and pure compound. Using electronic and fluorescence microscopy, along with bacterial mutant strains with marked divisional septum, the membrane was defined as the primary target of Fluopsin C in the tested bacteria.

Fluopsin C for Treating Multidrug-Resistant Infections: In vitro Activity Against Clinically Important Strains and in vivo Efficacy Against Carbapenemase-Producing Klebsiella pneumoniae.

The increasing emergence of multidrug-resistant (MDR) organisms in hospital infections is causing a global public health crisis. The development of drugs with effective antibiotic action against such agents is of the highest priority. In the present study, the action of Fluopsin C against MDR clinical isolates was evaluated under in vitro and in vivo conditions. Fluopsin C was produced in cell suspension culture of Pseudomonas aeruginosa LV strain, purified by liquid adsorption chromatography and identified by mass spectrometric analysis. Bioactivity, bacterial resistance development risk against clinically important pathogenic strains and toxicity in mammalian cell were initially determined by in vitro models. In vivo toxicity was evaluated in Tenebrio molitor larvae and mice. The therapeutic efficacy of intravenous Fluopsin C administration was evaluated in a murine model of Klebsiella pneumoniae (KPC) acute sepsis, using six different treatments. The in vitro results indicated MIC and MBC below 2 µg/mL and low bacterial resistance development frequency. Electron microscopy showed that Fluopsin C may have altered the exopolysaccharide matrix and caused disruption of the cell wall of MDR bacteria. Best therapeutic results were achieved in mice treated with a single dose of 2 mg/kg and in mice treated with two doses of 1 mg/kg, 8 h apart. Furthermore, acute and chronic histopathological studies demonstrated absent nephrotoxicity and moderate hepatotoxicity. The results demonstrated the efficacy of Fluopsin C against MDR organisms in in vitro and in vivo models, and hence it can be a novel therapeutic agent for the control of severe MDR infections.

Molecular identification and histological aspects of Renicola sloanei (Digenea: Renicolidae) in Puffinus puffinus (Procellariiformes): a first record.

Renicolids are parasites that inhabit the renal tubules and ureters of molluscivorous and piscivorous birds. Puffinus puffinus is a migratory seabird that was identified as the definitive host of Renicola spp. Studies focusing on the renicolid species and the resulting renal lesions are valuable for their association with causes of stranding in seabirds. The aim of this study was to identify the renicolid trematodes and evaluate the histological findings in two P. puffinus stranded on the coast of Paraná state, Brazil. The parasites were evaluated by histologic, ultrastructural and molecular assays, while tissue changes were analyzed by histologic methods. The morphological and morphometrical characteristics of the parasites, along with polymerase chain reaction and sequencing assays (ribosomal and mitochondrial regions), identified the species as Renicola sloanei. The results also suggest that this helminth can be the adult form of Cercaria pythionike. The dilation of collecting ducts was the main histological finding in the kidneys. In conclusion, R. sloanei was identified, and for the first time, P. puffinus was described as a host of this digenean inducing mild renal changes.

Molecular identification and histological aspects of Renicola sloanei (Digenea: Renicolidae) in Puffinus puffinus (Procellariiformes): a first record / Identificação molecular e aspectos histológicos de Renicola sloanei (Digenea: Renicolidae) em Puffinus puffinus (Procellariiformes): primeiros registros

Abstract Renicolids are parasites that inhabit the renal tubules and ureters of molluscivorous and piscivorous birds. Puffinus puffinus is a migratory seabird that was identified as the definitive host of Renicola spp. Studies focusing on the renicolid species and the resulting renal lesions are valuable for their association with causes of stranding in seabirds. The aim of this study was to identify the renicolid trematodes and evaluate the histological findings in two P. puffinus stranded on the coast of Paraná state, Brazil. The parasites were evaluated by histologic, ultrastructural and molecular assays, while tissue changes were analyzed by histologic methods. The morphological and morphometrical characteristics of the parasites, along with polymerase chain reaction and sequencing assays (ribosomal and mitochondrial regions), identified the species as Renicola sloanei. The results also suggest that this helminth can be the adult form of Cercaria pythionike. The dilation of collecting ducts was the main histological finding in the kidneys. In conclusion, R. sloanei was identified, and for the first time, P. puffinus was described as a host of this digenean inducing mild renal changes.

Resumo Renicolídeos são parasitos que habitam túbulos renais e ureteres de aves que se alimentam de moluscos e peixes. Puffinus puffinus, ave marinha migratória, foi registrada como hospedeiro definitivo de Renicola spp. Estudos relacionados com as espécies de renicolídeos e as lesões renais resultantes são importantes para o entendimento das causas de óbito de aves marinhas. O objetivo deste estudo foi identificar os trematódeos renicolídeos e avaliar os achados histológicos em dois P. puffinus encalhados no litoral do Estado do Paraná, Brasil. Os parasitos foram avaliados por ensaios histológicos, ultraestruturais e moleculares, enquanto as alterações teciduais foram analisadas por métodos histológicos. As características morfológicas e morfométricas dos parasitos, juntamente com a reação em cadeia da polimerase e sequenciamento (regiões ribossomal e mitocondrial), identificaram a espécie como Renicola sloanei. Os resultados também sugerem que este helminto pode ser a forma adulta de Cercaria pythionike. A dilatação dos ductos coletores foi o principal achado histológico renal. Em conclusão, R. sloanei foi identificado, e pela primeira vez P. puffinus foi descrito como hospedeiro deste digenético induzindo alterações renais discretas.

RNAm expression profile of cancer marker genes in HepG2 cells treated with different concentrations of a new indolin-3-one from Pseudomonas aeruginosa.

The present study tested the effects of a newly identified indolin-3-one compound (compound 1), produced by Pseudomonas aeruginosa, on HepG2 cells. The MTT assays demonstrated decreased metabolic activities in HepG2 cells treated with compound 1, with dose- and time-dependent intensifying effect, starting at a concentration of 40 µM. The IC50 after 24, 48, 72, and 96 h treatments were 41.35, 52.7, 92.79 and 66.65 µM of compound 1, respectively. Below 80 µM, no significative damage on erythrocytes membranes was observed by the hemolytic assays. The RT-qPCR revealed that the compound modulated key genes involved in carcinogenesis process, indicating possible indolin-3-one mechanisms of action. The data showed that gene expression alterations promoted by compound 1, in concentrations up to 60 µM after 48 h, led to a decrease in cellular progression and there was no direct cellular damage. In addition, non-cytotoxic concentrations of compound 1 halved the concentration of the chemotherapeutic doxorubicin, maintaining similar therapeutic effect against HepG2 cells. The novelty of the molecule and the biological activities observed in the present study emphasize the potential of the compound 1 in cancer therapy research.

Prevalence of Eimeria spp. in calves from dairy farms in northern Paraná state, Brazil.

Bovine coccidiosis is a disease of major importance in cattle herds across the world. The disorder mainly affects young calves, and E. bovis and E. zuernii are considered the most pathogenic species of the genus, however, E. alabamensis have been described in grazing calves. In this study, the prevalence of Eimeria spp. was evaluated in calves on dairy farms in the northern region of the state of Paraná, Brazil. Four hundred calves on 44 dairy farms were tested for the presence of coccidian oocysts. The positives were re-examined and the oocysts were morphometrically analyzed for species identification. All the farms were contaminated and 205 animals (51.25%) presented Eimeria spp. oocysts. Among these, 146 animals (71.22%) were co-infected by two or more species of coccidia. Ten species of Eimeria were identified: E. bovis (in 30.25% of the positive samples), E. alabamensis (26.75%), E. zuernii (22.00%), E. ellipsoidalis (18.50%), E. auburnensis (13.75%), E. canadensis (8.00%), E. cylindrica (7.25%), E. subspherica (5.00%), E. bukidnonensis (3.00%) and E. brasiliensis (0.75%). This study demonstrates the high prevalence of Eimeria spp. in the northern region of Paraná, Brazil, and detection for the first time in our region the pathogenic species E. alabamensis.

Prevalence of Eimeria spp. in calves from dairy farms in northern Paraná state, Brazil / Prevalência de Eimeria spp. em bezerros de propriedades leiteiras do norte do estado do Paraná, Brasil

Abstract Bovine coccidiosis is a disease of major importance in cattle herds across the world. The disorder mainly affects young calves, and E. bovis and E. zuernii are considered the most pathogenic species of the genus, however, E. alabamensis have been described in grazing calves. In this study, the prevalence of Eimeria spp. was evaluated in calves on dairy farms in the northern region of the state of Paraná, Brazil. Four hundred calves on 44 dairy farms were tested for the presence of coccidian oocysts. The positives were re-examined and the oocysts were morphometrically analyzed for species identification. All the farms were contaminated and 205 animals (51.25%) presented Eimeria spp. oocysts. Among these, 146 animals (71.22%) were co-infected by two or more species of coccidia. Ten species of Eimeria were identified: E. bovis (in 30.25% of the positive samples), E. alabamensis (26.75%), E. zuernii (22.00%), E. ellipsoidalis (18.50%), E. auburnensis (13.75%), E. canadensis (8.00%), E. cylindrica (7.25%), E. subspherica (5.00%), E. bukidnonensis (3.00%) and E. brasiliensis (0.75%). This study demonstrates the high prevalence of Eimeria spp. in the northern region of Paraná, Brazil, and detection for the first time in our region the pathogenic species E. alabamensis.

Resumo A coccidiose bovina é uma doença de grande importância em rebanhos ao redor do mundo. A desordem afeta principalmente bezerros jovens, e E. bovis e E. zuernii consideradas as espécies mais patogênicas deste gênero, causando grave enterite em animais infectados. No entanto, casos de E. alabamensis foram descritos em bezerros mantidos a pasto. No presente estudo, a prevalência de Eimeria spp. foi avaliada em bezerros de gado leiteiro da região norte do estado do Paraná, Brasil. Quatrocentos bezerros foram amostrados e testados para a presença de oocistos de coccídios. Os positivos foram re-examinados e os oocistos analisados morfologicamente para identificação da espécie. Todas as fazendas estavam contaminadas e 205 (51,25%) animais apresentaram oocistos de Eimeria spp. Destes, 146 (71,22%) animais estava co-infectados por duas ou mais espécies de coccídio. Dez espécies de Eimeria foram identificadas: E. bovis (30,25% de amostras positivas), E. alabamensis (26,75%), E. zuernii (22,00%), E. ellipsoidalis (18,50%), E. auburnensis (13,75%), E. canadensis (8,10%), E. cylindrica (8,00%), E. subspherica (5,00%), E. bukidnonensis (3,00%) e E. brasiliensis (0,75%). Este estudo demonstra a alta prevalência de Eimeria spp. na região norte do estado do Paraná, Brasil, e a detecção, pela primeira vez, de E. alabamensis.

Standardisation of egg-viability assays for Fasciola hepatica and Calicophoron daubneyi: A tool for evaluating new technologies of parasite control.

Fasciola hepatica and Calicophoron daubneyi, liver and rumen flukes respectively, infect ruminants throughout Europe. There is considerable interest in the development of vaccines and in testing new potential anthelmintic agents against these species. One potential target of new control measures is the parasite egg, as interference at this stage of the life cycle could aid in blocking the transmission of infection, and some experimental vaccines have been shown to affect egg viability. In this study, we describe the standardisation of protocols to evaluate the viability of eggs of these two parasites. Eggs were recovered from adult parasites collected in a commercial abattoir, from naturally infected cattle. A protocol for in vitro development of F. hepatica eggs was optimised based on previously published methods, with variations in duration and temperature of incubation. A new protocol for measurement of rumen fluke egg development in vitro was designed, based on testing different temperatures and periods of incubation, with or without light exposure. The protocols described here may be used in the future for comparing experimental groups when new technologies for parasite control are tested. In addition, the methods described for C. daubneyi present new information on the biology of this parasite.

Pathogenicity of Nc-Bahia and Nc-1 strains of Neospora caninum in experimentally infected cows and buffaloes in early pregnancy.

Neospora caninum is a protozoan parasite known as an important cause of bovine abortion worldwide. Little is currently known about how different strains of N. caninum vary in their pathogenicity. In this study, we compared a Brazilian strain, Nc-Bahia, with the first isolate of this coccidian, Nc-1. Eight cows and seven buffaloes were submitted to fixed-time artificial insemination protocols for a better control of pregnancy. Group 1 was inoculated with Nc-Bahia (n = 8; five cows and three buffaloes), and Group 2 was inoculated with Nc-1 (n = 5; two cows and three buffaloes). One nonpregnant female of each species was left uninfected as sentinel controls for potential environmental infection. All inoculated animals received 5 × 10(8) tachyzoites of N. caninum, by intravenous route, on the 70th day of gestation. Uninfected animals remained seronegative throughout the experiment, indicating no exogenous infection, whereas all inoculated animals became seropositive to N. caninum. In Group 1, abortion was found in only one cow on 42 days postinfection (dpi; frequency of abortion = 12.5%), whilst all animals from Group 2 aborted on 35 dpi (frequency of abortion = 100%). Parasite DNA was detected by seminested PCR in maternal, foetal and placental tissues, confirming vertical transmission in Groups 1 and 2, although histological lesions had different frequencies and degrees of severity between the groups. There was evidence of lower pathogenicity of Nc-Bahia compared to Nc-1 when used in experimental infection, as it caused fewer abortions, as well as less frequent and milder histological lesions. This was the first time Nc-Bahia has been used for experimental infection.

Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis.

The aim of this study was to optimize a PCR assay that amplifies an 843 pb fragment from the p28 gene of Ehrlichia canis and compare it with two other PCR methods used to amplify portions of the 16S rRNA and dsb genes of Ehrlichia. Blood samples were collected from dogs suspected of having a positive diagnosis for canine ehrlichiosis. Amplification of the p28 gene by PCR produced an 843-bp fragment and this assay could detect DNA from one gene copy among 1 billion cells. All positive samples detected by the p28-based PCR were also positive by the 16S rRNA nested-PCR and also by the dsb-based PCR. Among the p28-based PCR negative samples, 55.3% were co-negatives, but 27.6% were positive in 16S rRNA and dsb based PCR assays. The p28-based PCR seems to be a useful test for the molecular detection of E. canis, however improvements in this PCR sensitivity are desired, so that it can become an important alternative in the diagnosis of canine ehrlichiosis.

Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis / Avaliação da sensibilidade da PCR em uma etapa com base no gene p28 de Ehrlichia canis e sua aplicação no diagnóstico da erliquiose canina

The aim of this study was to optimize a PCR assay that amplifies an 843 pb fragment from the p28 gene of Ehrlichia canis and compare it with two other PCR methods used to amplify portions of the 16S rRNA and dsb genes of Ehrlichia. Blood samples were collected from dogs suspected of having a positive diagnosis for canine ehrlichiosis. Amplification of the p28 gene by PCR produced an 843-bp fragment and this assay could detect DNA from one gene copy among 1 billion cells. All positive samples detected by the p28-based PCR were also positive by the 16S rRNA nested-PCR and also by the dsb-based PCR. Among the p28-based PCR negative samples, 55.3 percent were co-negatives, but 27.6 percent were positive in 16S rRNA and dsb based PCR assays. The p28-based PCR seems to be a useful test for the molecular detection of E. canis, however improvements in this PCR sensitivity are desired, so that it can become an important alternative in the diagnosis of canine ehrlichiosis.

O objetivo deste estudo foi aperfeiçoar um ensaio de PCR que amplificasse um fragmento de 843 pares de bases do gene p28 da Ehrlichia canis e compará-lo com outros dois métodos de PCR utilizados para amplificar partes do gene 16S rRNA e dsb do gênero Ehrlichia. Amostras sanguíneas foram colhidas de cães com diagnóstico clínico de erliquiose. A amplificação do gene p28 pela PCR produziu um fragmento de 843pb e esse ensaio permitiu a detecção do DNA de um parasita dentre 1 bilhão de células. Todas as amostras positivas detectadas pela PCR baseada no gene p28 foram também positivas pela nested PCR para detecção do gene 16S rRNA e também pela PCR dsb. Dentre as amostras negativas para a PCR p28, 55,3 por cento foram co-negativas, mas 27,6 por cento foram positivas pela PCR baseada nos genes 16S rRNA e dsb. A PCR p28 parece ser um teste útil para detecção molecular de E. canis, entretanto otimizações na sensibilidade nesta PCR são necessárias, para que esta técnica se torne uma importante alternativa no diagnóstico da erliquiose canina.