Molecular identification and antimicrobial resistance pattern of Nocardia isolated from 14 diseased dogs and cats.

Nocardia are ubiquitous, saprophytic and opportunistic bacteria. They cause a set of pyogenic clinical infections in animals and humans, particularly immunocompromised patients, mostly affecting the skin and respiratory tract, with refractoriness to conventional therapy. The most descriptions of nocardial infections in companion animals involve case reports, and there are scarce case series studies focused on canine and feline nocardiosis in which diagnosis has been based on molecular techniques. We investigated epidemiological aspects, clinical findings, in vitro susceptibility profile, and molecular identification of Nocardia using PCR-based method targeted 16S rRNA gene in twelve dogs and two cats. Among dogs were observed cutaneous lesions (8/12 = 67%), pneumonia (3/12 = 25%), and encephalitis (2/12 = 17%), whereas cats developed cutaneous lesions and osteomyelitis. Nocardia and canine morbillivirus coinfection was described in six dogs (6/12 = 50%). A high mortality rate (6/8 = 75%) was seen among dogs. Three dogs (3/4 = 75%) and one cat (1/2 = 50%) with systemic signs (pneumonia, encephalitis, osteomyelitis), and 83% (5/6) of dogs with a history of concomitant morbillivirus infection died. N. nova (5/12 = 42%), N. cyriacigeorgica (3/12 = 25%), N. farcinica (2/12 = 17%), N. veterana (1/12 = 8%), and N. asteroides (1/12 = 8%) species were identified in dogs, whereas N. africana and N. veterana in cats. Among the isolates from dogs, cefuroxime (12/12 = 100%), amikacin (10/12 = 83%), gentamycin (10/12 = 83%), and imipenem (10/12 = 83%) were the most effective antimicrobials, whereas cefuroxime, cephalexin, amoxicillin/clavulanic acid, imipenem, and gentamycin were efficient against isolates from cats. Multidrug resistance was observed in 36% (5/14) of isolates. We describe a variety of Nocardia species infecting dogs and cats, multidrug-resistant ones, and a high mortality rate, highlighting a poor prognosis of nocardiosis in companion animals, particularly among animals systemically compromised or coinfected by canine morbillivirus. Our study contributes to species identification, in vitro antimicrobial susceptibility profile, clinical-epidemiological aspects, and outcome of natural Nocardia-acquired infections in dogs and cats.

Culturable Microbial Population From the Upper Respiratory Tract of 1,010 Clinically Healthy Horses in Southern Brazil.

Microbiological diagnosis of equine respiratory infections is essential for disease management. However, reliable diagnosis can be a challenge due to colonization of the upper respiratory tract (URT) by a diverse microbial population, and because there is a lack of studies with samples from healthy animals. Aiming to guide adequate URT culture, this work reports culturable microbial population from the URT of 1,010 apparently healthy horses from 341 farms in Southern Brazil and identifies the putative presence of pathogenic microorganisms. Nasal swabs were cultured in 5% blood agar, and the isolates were phenotypically identified to genus level. A diverse respiratory microbial population was found, mostly composed of Gram-positive bacteria, including Staphylococcus spp., Bacillus spp., Streptococcus spp. and Corynebacterium spp. The microbiological profile from the nasal cavity of 911 horses was described, with the five most common profiles being: (1) Staphylococcus sp. + Gram-negative bacilli (12.67%), (2) Staphylococcus sp. in pure culture (12.47%), (3) Staphylococcus sp. + Bacillus sp. (10.10%), (4) Gram-negative bacilli in pure culture (6.93%), and (5) Staphylococcus sp. + Bacillus sp. + Gram-negative bacilli (6.73%). Streptococcus equi equi and Rhodococcus equi were detected in 34 horses (3.37%), demonstrating the presence of pathogenic bacteria along with commensal microorganisms in apparently healthy animals. The disclosed data may guide and facilitate microbiological diagnosis of URT infection in horses. The significant presence of Gram-negative bacilli was evidenced, as well as the occurrence of relevant pathogens, such as S. equi equi and R. equi, thus helping to improve diagnosis and antimicrobial therapy.

GAPDH, rhbC, and vapA gene expression in Rhodococcus equi cultured under different iron concentrations.

The ability of Rhodococcus equi to survive in macrophages and cause pneumonia in foals depends on vapA and rhbC genes, which produce the virulence-associated protein A (VapA) and the rhequichelin siderophore, respectively. Virulent R. equi acquires Fe from transferrin by unknown mechanisms. Our objectives were to determine the role of GAPDH in Fe homeostasis, to further characterize GAPDH, rhbC, and vapA expression under iron homeostasis, and to document the occurrence of rhbC gene in R. equi isolates. Therefore, vapA + R. equi was cultured under excessive, physiologic, and restricted iron concentrations, and quantitative culture and gene expression were performed. The relative expression of GAPDH, rhbC, and vapA after 48 h of culture were analyzed by qPCR. To determine the rhbC occurrence, total DNA was extracted from R. equi isolated from foals with clinical rhodococcosis (n = 22), healthy horses (feces, n = 16; nasal swab, n = 9), soil (n = 6), and 2 ATCC reference strains. Conventional PCR was performed to identify genus/species, vapA, and rhbC genes. Iron restriction proportionally decreased R. equi growth rates, and induced high expression of both GAPDH and vapA. The putative role of GAPDH in R. equi iron homeostasis should be further investigated. rhbC was significantly up-regulated under both Fe excess and critical starvation. The rhbC gene was identified in all clinical isolates and soil, but it was absent in 2 isolates from healthy horses, suggesting that rhequichelin is not required for R. equi nasal and intestinal colonization.

Genomic comparison of Clostridium chauvoei isolates from classical and visceral clinical manifestation.

Clostridium chauvoei is the etiological agent of blackleg, an infectious disease affecting cattle and small ruminants worldwide. This disease can manifest as classical blackleg, a condition in which skeletal muscles are affected and visceral blackleg, which affects the heart, sublingual muscles, and the diaphragm. The pathogenesis of the visceral form of the disease is poorly understood. The objective of this study is to determine and analyze complete genomic sequences of six C. chauvoei strains, five isolates from skeletal muscle and one isolate from a visceral case of blackleg in Brazil, to provide insights into the differences in pathogenic profiles of strains causing the different forms of disease. The full genomes of the six C. chauvoei strains were sequenced and comparative analyses were performed among these genomes and the C. chauvoei reference strain JF4335. The results of this study revealed that the genomes of the C. chauvoei strains analyzed are highly conserved; no particular differences were noted that could be associated with the two different clinical manifestations of the disease.

The survival and hepatic and muscle glucose and lactate levels of Rhamdia quelen inoculated with Aeromonas hydrophila and treated with terpinen-4-ol, carvacrol or thymol.

The aim of this study was to evaluate the resistance of Rhamdia quelen (silver catfish) to Aeromonas hydrophila infection after treatment with pure and nanoencapsulated forms either terpinen-4-ol, thymol, or carvacrol and the effects of these treatments on fish metabolic responses. After A. hydrophila inoculation, fish were treated with 30 min daily baths for 6 consecutive days with terpinen-4-ol, thymol, or carvacrol in their pure or nanoencapsulated forms at concentrations of 5, 10, 15 or 25 mg L-1. A positive control group, negative control group and saline group were also included. Survival was evaluated at the end of treatment for six consecutive days. Muscle and liver were collected to determine glucose and lactate levels. The fish treated with the nanoencapsulated form of the compounds had a high survival rate, similar to saline group and negative control groups. The carvacrol, thymol and terpinen-4-ol nanoencapsulated forms improved survival of silver catfish infected with A. hydrophila. Muscle and liver glucose and lactate levels are not indicated as biomarkers because they did not present any correlation between the metabolic state of the fish and the bacterial infection.

Antigenic characterization of Moraxella bovis, Moraxella bovoculi and Moraxella ovis strains with potential use in vaccines.

Moraxella bovis is historically known as the primary agent of infectious bovine keratoconjunctivitis (IBK). However, Moraxella bovoculi and Moraxella ovis are also reported to be involved in the pathogenesis of IBK, therefore, these three species should be included in the development of a new vaccine with a broad-spectrum protection against the disease natural challenge. In this study we investigated the antigenic properties of clinical isolates and reference strains of M. bovis, M. bovoculi and M. ovis using a novel in vitro approach for vaccine evaluation based on two techniques, flow cytometry and western blotting (WB). Here, we demonstrated that rabbit antisera produced against reference M. bovis strain and commercial bacterin showed low number of IgG with capacity to recognize a panel of heterologous strains composed by M. bovoculi and M. ovis. On the other hand, the antisera generated against two clinical isolates of M. ovis (Mov2 and Mov3) presented high cross-reactivity levels against all M. ovis and M. bovis strains evaluated. Similarly, the antisera against Mbv3 (clinical isolate of M. bovoculi) had high levels of IgG associated on the surface of all M. bovoculi strains and most of the M. ovis strains analyzed. The WB analysis demonstrated that Moraxella spp. has multiple immunogenic antigens and most of them are shared between the three species. Based on the cross-reactivity analysis and considering the relative number of IgGs associated on the bacterial surface, we suggest that a multivalent vaccine including Mbv3, Mov2 and Mov3 strains may provide a strong and broad protection against all strains involved in IBK outbreaks.

Chloroquine inhibits Rhodococcus equi replication in murine and foal alveolar macrophages by iron-starvation.

Rhodococcus equi preferentially infects macrophages causing pyogranulomatous pneumonia in young foals. Both the vapA and rhbC genes are up-regulated in an iron (Fe)-deprived environment, such as that found within macrophages. Chloroquine (CQ) is a drug widely used against malaria that suppresses the intracellular availability of Fe in eukaryotic cells. The main objective of this study was to evaluate the ability of CQ to inhibit replication of virulent R. equi within murine (J774A.1) and foal alveolar macrophages (AMs) and to verify whether the mechanism of inhibition could be Fe-deprivation-dependent. CQ effect on R. equi extracellular survival and toxicity to J774A.1 were evaluated. R. equi survival within J774A.1 and foal AMs was evaluated under CQ (10 and 20µM), bovine saturated transferrin (bHTF), and bovine unsaturated transferrin (bATF) exposure. To explore the action mechanism of CQ, the superoxide anion production, the lysozyme activity, as well as the relative mRNA expression of vapA and rhbC were examined. CQ at≤20µM had no effect on R. equi extracellular multiplication and J774A.1 viability. Exposure to CQ significantly and markedly reduced survival of R. equi within J774A.1 and foal AMs. Treatment with bHTF did not reverse CQ effect on R. equi. Exposure to CQ did not affected superoxide anion production or lysozyme activity, however vapA and rhbC expression was significantly increased. Our results reinforce the hypothesis that intracellular availability of Fe is required for R. equi survival, and our initial hypothesis that CQ can limit replication of R. equi in J774A.1 and foal AMs, most likely by Fe starvation.

Prevalence of Streptococcus equi subsp. equi in horses and associated risk factors in the State of Rio Grande do Sul, Brazil.

The aim of this study was to estimate the prevalence of equine strangles and to identify associated risk factors for this disease through a cross-sectional study of nasal swabs. Nasal swabs (n=1010) from healthy equines (absence of nasal discharge, lymphadenopathy and cough) from 341 farms were plated on 5% blood agar; of these horses, 24 were identified as positive for Streptococcus equi through isolation, PCR and DNA sequencing. The estimated prevalence for individual animals was 2.3%, and for herds, it was 5.86%. Statistical analysis identified the following as associated risk factors: the number of group events that were attended by the equines (PR: 1.06); the sharing of food containers (PR: 3.74); and at least one previous positive diagnosis of strangles on the farm (PR: 3.20). These results constitute an epidemiological contribution to the horse industry and may support measures for the future control of the disease.

Differences in the antimicrobial susceptibility profiles of Moraxella bovis, M. bovoculi and M. ovis.

The aim of this study was to determine the differences in the antimicrobial susceptibility profiles of Moraxella bovis, M. bovoculi and M. ovis. Thirty-two strains of Moraxella spp. isolated from cattle and sheep with infectious keratoconjunctivitis were tested via broth microdilution method to determine their susceptibility to ampicillin, cefoperazone, ceftiofur, cloxacillin, enrofloxacin, florfenicol, gentamicin, neomycin, oxytetracycline and penicillin. The results demonstrated that Moraxella spp. strains could be considered sensitive for most of the antimicrobials tested in this study, but differences between the antimicrobial susceptibility profiles of these three Moraxella species were found. M. bovis might differ from other species due to the higher MIC and MBC values it presented.

Phylogenetic analysis and genetic diversity of 3' region of rtxA gene from geographically diverse strains of Moraxella bovis, Moraxella bovoculi and Moraxella ovis.

The cytotoxin A (MbxA) is one of the main virulence factors of Moraxella bovis involved in the pathogenesis of infectious bovine keratoconjunctivitis (IBK). Moraxella ovis and Moraxella bovoculi, suspected to be associated with infectious keratitis in sheep and cattle respectively, also have a gene that encodes the cytotoxin A (movA and mbvA, respectively). The aim of this study was to determine the molecular sequence of the 3' region of the cytotoxin gene of Moraxella spp. strains isolated from clinical cases to establish phylogenetic and evolutionary comparisons. PCR amplification, nucleotide sequencing (nt) and amino acid (aa) sequence prediction were performed, followed by the sequences comparison, identity level calculation and selective pressure analysis. The phylogenetic reconstruction based on nt and aa sequences clearly differentiate M. bovis (n=15), M. bovoculi (n=11) and M. ovis (n=7) and their respective reference strains. An alignment of 843nt revealed high similarity within bacterial species (MbxA=99.9% nt and aa; MbvA=99.3% nt and 98.8% aa; MovA=99.5% nt and 99.3% aa). The similarity of partial sequences (nt 1807-2649) of MbxA in relation to MbvA and MovA ranged from 76.3 to 78.5%; similarity between MbvA and MovA ranged from 95.7 to 97.5%. A negative selection on mbvA and movA sequences was revealed by the molecular evolution analysis. The phylogenetic analysis of movA and mbvA allowed different strains of Moraxella spp. to be grouped according to the period of isolation. Sequence analysis of cytotoxin may provide insights into genetic and evolutionary relationships and into the genetic/molecular basis of Moraxella spp.

Genotypic and phenotypic detection of efflux pump in Rhodococcus equi.

The req_39680 gene, associated to a putative efflux system, was detected in 60% (54/90) of R. equi isolates by PCR. The phenotypic expression of efflux mechanism was verified in 20% of the isolates using ethidium bromide. For the first time, the expression of efflux mechanism was demonstrated in R. equi.

Acute myonecrosis in horse caused by Clostridium novyi type A.

The objective of this study was to describe the first report involving a case of equine acute myonecrosis caused by C. novyi type A with an emphasis on clinical signs, the pathological and bacteriological analysis, and molecular identification of the microorganisms as the key of the definitive diagnosis.

The use of eugenol against Aeromonas hydrophila and its effect on hematological and immunological parameters in silver catfish (Rhamdia quelen).

The aim of this study was to evaluate the activity of eugenol against the fish pathogen Aeromonas hydrophila and eugenol's effect on hematological and natural immune parameters in silver catfish (Rhamdia quelen). In vitro, eugenol showed weak activity against A. hydrophila, but in vivo, at a subinhibitory concentration (10 mg L(-1)), it promoted survival in infected silver catfish. Eugenol (50 µg mL(-1)) reduced the hemolytic activity of A. hydrophila supernatant in vitro in fish erythrocytes. Subjecting catfish to eugenol baths (5 and 10 mg L(-1)) for five days did not alter the hematological and immunological parameters studied in this work. Based on these results, eugenol can be used to treat or prevent bacterial diseases in fish.

Bronchopneumonia in wild boar (Sus scrofa) caused by Rhodococcus equi carrying the VapB type 8 plasmid.

BACKGROUND: Rhodococcus equi is associated with pyogranulomatous infections, especially in foals, and this bacterium has also emerged as a pathogen for humans, particularly immunocompromised patients. R. equi infections in pigs, wild boar (Sus scrofa) and humans are mainly due to strains carrying the intermediate virulence (VapB) plasmid. In Brazil, R. equi carrying the VapB type 8 plasmid is the most common type recovered from humans co-infected with the human immunodeficiency virus (HIV). R. equi infection in pigs and wild boar is restricted predominantly to the lymphatic system, without any reports of pulmonary manifestations. FINDINGS: This report describes the microbiological and histopathological findings, and molecular characterization of R. equi in two bronchopneumonia cases in wild boar using PCR and plasmid profile analysis by digestion with restriction endonucleases. The histological findings were suggestive of pyogranulomatous infection, and the plasmid profile of both R. equi isolates enabled the characterization of the strains as VapB type 8. CONCLUSIONS: This is the first report of bronchopneumonia in wild boar due to R. equi. The detection of the VapB type 8 plasmid in R. equi isolates emphasize that wild boar may be a potential source of pathogenic R. equi strains for humans.

Diversity of seM in Streptococcus equi subsp. equi isolated from strangles outbreaks.

Strangles is the main upper respiratory tract disease of horses. There are currently no studies on the changes in alleles of the M protein gene (seM) in Brazilian isolates of Streptococcus equi ssp. equi (S. equi). This study aimed to analyze and differentiate molecularly S. equi isolates from equine clinical specimens from southern Brazil, between 1994 and 2010. seM alleles were analyzed in 47 isolates of S. equi obtained from clinical cases of strangles (15 Thoroughbred horses, 29 Crioulo breed horses and three Brasileiro de Hipismo--BH). seM alleles characterization was performed by comparing variable region sequences of the seM gene. The alleles were also phylogenetically grouped by Neighbor-joining analysis, which demonstrated the geographic distribution of those in properties from southern Brazil. Fifteen alleles of the gene seM were found among the 47 S. equi isolates analyzed. Among these, only one allele (seM-61), which was identified in seven isolates (14.9%), was found in the database PubMLST-seM. Within the new alleles, allele seM-115 was the most prevalent, having been found in 13 isolates (27.7%), followed by allele seM-117 in 10 isolates (21.3%). In the Brazilian horse population studied, there is greater diversity of M protein alleles in S. equi isolates compared to worldwide data deposited in PubMLST-seM. Among the 15 seM alleles identified, only one allele sequence was previously published. The alleles identification is important to control the disease by guiding selection of strains for the manufacture of commercial and autogenous vaccines.

Short report: Identification of virulence-associated plasmids in Rhodococcus equi in humans with and without acquired immunodeficiency syndrome in Brazil.

Virulence of Rhodococcus equi strains from 20 humans in Brazil was investigated by using a polymerase chain reaction to characterize isolates as virulent (VapA), intermediately virulent (VapB), and avirulent. Nine isolates were obtained from human immunodeficiency virus (HIV)-positive patients, six from HIV-negative patients, and five from patients of unknown status. Five isolates were VapB positive, four were VapA positive, and eleven were avirulent. Among the nine isolates from HIV-positive patients, five contained VapB plasmids and two contained VapA plasmids. Five VapB-positive isolates had the type 8 virulence plasmid. Eleven of the patients had a history of contact with livestock and/or a farm environment, and none had contact with pigs.

Antimicrobial activity of propolis extract against Staphylococcus coagulase positive and Malassezia pachydermatis of canine otitis.

The aims of this study were to evaluate the antimicrobial potential of propolis extract by determining the minimum bactericidal concentration (MBC) for coagulase-positive Staphylococcus isolates (Staphylococcus aureus and Staphylococcus intermedius) and the minimum fungicidal concentration (MFC) for Malassezia pachydermatis isolates. The microorganisms were assayed using broth microdilution techniques. The MBC(90) was 21 mg mL(-1), and the MFC(90) was 5.3 mg mL(-1). The propolis extract was found to exhibit antimicrobial activity against both pathogens.

Molecular characterization of Rhodococcus equi Isolates of horse breeding farms from an endemic region in South of Brazil by multiplex PCR.

Rhodococcus equi is a gram-positive coco-bacillus and an intracellular opportunistic pathogen which causes pneumonia in foals. It is widely detected in environment and has been isolated from several sources, as soil, feces and gut from health and sick foals. The goal of this study was to characterize the epidemiological status (endemic, sporadic or no infection) of horse breeding farms from Bage County in South of Brazil, using a multiplex PCR. One hundred and eighteen R. equi isolates were identified by biochemical tests and submitted to a specie-specific and vapA multiplex PCR. These isolates were obtained from: three farms where the R. equi infection has been noticed, two farms where the disease has been not reported and one farm where the disease is frequent. All clinical isolates from horse breeding farms where the disease is endemic and/or sporadic were vapA-positive. None environmental isolates were vapA-positive. In three horse breeding farms with sporadic R. equi infection, 11.54% of the isolates from adult horse feces were vapA-positive. The multiplex PCR technique has proven to be effective for the molecular and epidemiological characterization of the R. equi isolates in horse breeding farms. An important finding in this study was the isolation of vapApositive R. equi from adult horse feces, which is an evidence for other routes of dissemination of this pathogen in the farms.

Virulence factors and antimicrobial resistance of escherichia coli isolated from urinary tract of swine in southern of Brazil.

The present study determined the molecular and resistance patterns of E. coli isolates from urinary tract of swine in Southern of Brazil. Molecular characterization of urinary vesicle samples was performed by PCR detection of virulence factors from ETEC, STEC and UPEC. From a total of 82 E. coli isolates, 34 (38.63%) harbored one or more virulence factors. The frequency of virulence factors genes detected by PCR were: pap (10.97%), hlyA (10.97%), iha (9.75%), lt (8.53%), sta (7.31%) sfa (6.09%), f4 (4.87%), f5 (4.87%), stb (4.87%), f6 (1.21%) and f41 (1.21%). Isolates were resistant to penicillin (95.12%), lincomycin (93.9%), erythromycin (92.68%), tetracycline (90.24%), amoxicillin (82.92%), ampicillin (74.39%), josamycin (79.26%), norfloxacin (58.53%), enrofloxacin (57.31%), gentamicin (39.02%), neomycin (37.8%), apramycin (30.48%), colistine (30.48%) and cefalexin (6.09%). A number of 32 (39.02%) E. coli isolates harbored plasmids.

Phenotypical assays and partial sequencing of the hsp60 gene for identification of Streptococcus equi.

Strangles is an acute and contagious disease characterized by inflammation of the upper respiratory tract of horses. The etiological agent of strangles is the bacteria S. equi subsp. equi, which belongs to the Lancefield group C. Opportunistic agents from the same group are frequently isolated from horses with strangles and may induce mistaken diagnoses. Among the subspecies of S. equi, the phenotypic features are almost undistinguishable; however, the pathogenic potential is widely differentiated. The aim of this study was to characterize S. equi isolates obtained from clinical samples of strangles by phenotypic tests and to analyze the partial sequences obtained from fragments of the hsp60 gene. In this work, 26 strains of Streptococcus spp. isolated from horse clinical samples were analyzed. By phenotypical assays, 18 were characterized as S. equi subsp. equi, five as S. equi subsp. zooepidemicus, two as S. dysgalactiae subsp. equisimilis, and one as Streptococcus sp. However 21 isolates were identified as S. equi subsp. equi and five as S. equi subsp. zooepidemicus by DNA sequencing. The sequencing of the partial hsp60 gene was demonstrated to be an alternative method to analyze and differentiate strains of Streptococcus spp. In addition, this method can be useful as a discriminatory tool for characterization of atypical isolates.