Urinary Electrophoretograms Performed by Capillary Electrophoresis: Comparison between Dogs and Cats.

Electrophoresis of urine to evaluate different urinary proteins has been used in recent years in veterinary medicine, as it can be a useful laboratory tool in the early detection of kidney damage. However, urinary capillary electrophoresis (UCE) has not been reported in healthy cats. In healthy dogs, reference intervals have been established and can be compared with pathological samples as these provide an easily interpretable pattern. The electrophoretogram in this study is divided into five fractions (F1-F5) by serum (albumin; alpha1-globulin; alpha2-globulin; beta-globulin; and gamma-globulin). Urine samples from 14 healthy cats were obtained by eco-guided cystocentesis. UCE was run in all samples and compared to 123 dog electrophoretograms from a previously published study. Fraction 2 (alpha1-globulin) was statistically decreased in cats (G1) compared to dogs (G2). Fraction 4 (beta-globulin) was statistically augmented in cats compared to the canine population (G2). Fraction 5 (gamma-globulin) was statistically decreased in cats (G1) compared to dogs (G2). No statistical correlation was found between each cat's serum and urinary fractions. The results of the present study suggest that UCE patterns in cats are similar to the ones described in dogs. UCE can be a non-invasive new diagnostic tool in cats as pathological patterns can be compared to normal ones.

Development of a specific polymerase chain reaction assay for the detection of Basidiobolus.

The etiology of chronic diarrhea is complex in humans and animals. It is always necessary to evaluate a list of differential diagnosis, including bacteria, protozoa and fungi. Basidiobolomycosis is a fungal disease reported sporadically worldwide, mainly caused by B. ranarum, a frequent organism found in soil or in the intestine and skin of lizards and frogs. It is an opportunistic pathogen that causes infections characterized by granulomatous lesions in the subcutaneous tissues as well as in the intestinal wall in humans and animals. In this work we have developed a PCR technique to differentiate Basidiobolus from other causes of intestinal disease in dogs and humans. To test the specificity of the PCR assay we included closely related organisms, common intestinal microbiota and pathogenic organisms, such as Aspergillus, Candida, Cryptosporidium, Escherichia, Giardia, Mucor, Proteus, Rhizopus and Salmonella. Pythium insidiosum, which cause clinically similar disease in dogs but require a different treatment. Only Basidiobolus was positive to the PCR assay.

Urinary phoretograms performed by capillary electrophoresis in dogs with chronic disease with or without Leishmania infantum infection.

Introduction: The study of early markers to detect kidney malfunction has increased in recent years since serum markers, such as creatinine increase when there is a 75% loss of renal mass. Urinary capillary electrophoresis (UCE) is an available laboratory technique that provides an easily interpretable electrophoretic pattern. This pattern in our study has been divided into five fractions as it is done in serum: fraction 1 migrating in the albumin zone, fraction 2 in the alpha1-globulins zone, fraction 3 in the alpha2-globulins zone, fraction 4 in the beta-globulins zone, and fraction 5 in the gamma globulins zone. UCE can be useful in the early diagnosis of renal disease. Material and methods: In this study, UCE was performed in dogs with azotemia and proteinuria due to chronic kidney disease (CKD) not related to Leishmania infantum (L. infantum) infection (G1, n = 11) and dogs with CKD related to L. infantum infection (G2, n = 17) and compared with reference intervals from healthy dogs (G0, n = 123), with the aim of comparing their phoretograms and assessing changes in the fractions of the phoretograms based on the health status of individuals. Results: Fraction 2 was statistically augmented in dogs with CKD (G1) when compared with the healthy population (G0) and dogs infected by L. infantum (G2). Fraction 3 was statistically increased in dogs with CKD (G1) and dogs infected by L. infantum (G2) compared with G0. Fraction 4 was found to be statistically decreased in dogs with CKD (G1) and dogs infected by L. infantum (G2) compared with G0. Fraction 5 was statistically higher in dogs with L. infantum (G2) compared with G0 and dogs with CKD (G1). No statistical relationship was found between the protein to creatinine ratio and different fractions from the urinary phoretogram in the study population. No statistical relationship was found between serum and urine fractions in the study population. Discussion: The results of the present study suggest that UCE is a promising non-invasive technique that might be used as a part of the diagnostic and follow-up in dogs with kidney disease due to different pathologies.

Evaluation of Urine Proteins by Capillary Electrophoresis.

Capillary electrophoresis (CE) is a laboratory method usually used to separate proteins in body fluids such as serum, cerebrospinal fluid, or urine. Separation of proteins in urine can have clinical applications for evaluating samples from healthy dogs and dogs with proteinuria in a qualitative way, which would not be possible with gel electrophoresis. Other advantages of CE over gel electrophoresis in serum include the reduced separation time (2 min vs. 20 min in a gel), reduction of waste harmful to humans and the environment, and ability to obtain a curve without the need for additional staining. This protocol is divided into four steps. Firstly, urine needs to be prepared prior to dialysis. Secondly, urine needs to undergo dialysis to eliminate compounds that could interfere with separation, and to concentrate the urine. The third step is CE using specific equipment. The last step is to separate the fractions of the phoretograms obtained in the previous step. This method is mostly an automatized process, easily reproducible, and that can be performed in any laboratory, as a part of the diagnostic or follow-up of patients with renal disease. Graphical abstract.

Reference intervals for electrophoretograms obtained by capillary electrophoresis of dialyzed urine from healthy dogs.

Electrophoresis of urine to evaluate protein fractions in dogs with proteinuria to differentiate glomerular from tubular damage has increased in recent years; however, capillary electrophoresis (CE) of urine has not been reported in a study of > 40 healthy animals, to our knowledge. We aimed to establish reference intervals (RIs) for the urine protein fractions obtained by CE of urine from healthy dogs. We obtained urine samples from 123 clinically healthy dogs of both sexes between December 2016 and April 2019; urine was frozen until CE was performed. The electrophoretic patterns obtained were divided into 5 protein fractions, and RIs were established in percentages and absolute values using nonparametric methods. RIs were obtained for the fractions (F) as follows: 5.5 to 56.2% for F1, 3.2 to 16.5% for F2, 3.5 to 16.2% for F3, 17.8 to 69.8% for F4, and 5.1 to 23.9% for F5. These RIs obtained by CE might be useful clinically as a basis for comparison with pathologic samples. Age was a statistically significant factor for F2 (p = 0.01) and F3 (p = 0.02), and sex was a statistically significant factor for F1 (p = 0.03).

[Prevalence of hepatitis E virus (HEV) and risk factors in pig workers and blood donors]. / Prevalencia del virus de la hepatitis E (VHE) y factores de riesgo en trabajadores de explotaciones porcinas y donantes voluntarios.

INTRODUCTION: Hepatitis E virus (HEV) is the main causative agent of enterically transmitted non-A non-B hepatitis with a self-limiting clinical presentation in humans. The increasing number of non-immigrant cases of hepatitis E and the high prevalence detected in pigs suggested that this species is a reservoir. Several studies have been published describing differences in the prevalence of anti-HEV antibodies between people exposed and not exposed to pigs, but the risk factors for the acquisition of the virus have not been well studied. The aim of this work was to study the prevalence of hepatitis E virus (HEV), IgG and IgM antibodies anti-HEV and the risk factors for the acquisition of this virus in a populations exposed and unexposed to pigs. METHODS: Sera from 212 people were tested for the presence of RNA-HEV, IgG and IgM antibodies anti-HEV. Positive results obtained using ELISA assays were confirmed by western-blot. RESULTS: RNA-HEV and IgM anti-VHE were not detected in any of the individuals. Twenty-five samples (11.8%) showed IgG anti-HEV, 21 (18.6%) persons in contact with pigs and 4 (4%) unexposed individuals (P=0.004); 19 (12.8%) had travelled out of Spain in the last year (P=0.507). None of the 25 seropositive people had consumed raw shellfish (P=0.999), whereas 23 (12.6%) had consumed raw vegetables (P=0.387) and 12 (30%) usually drank untreated water (P=0.008). CONCLUSION: These data support that HEV infection should be treated as an occupational disease illness in pig workers. Therefore, systematic application of hygiene measures in this group is highly recommended to avoid exposure to this virus.

Hepatitis E autochthonous infection in chronic liver disease.

Hepatitis E virus is endemic in many parts of the developing world and causes a self-limiting hepatitis in young adults, except in pregnant women and patients with chronic liver disease, where the mortality is high. Locally acquired hepatitis E is increasingly recognized in the developed world. It is caused by hepatitis E virus genotype 3, affects the middle-aged and the elderly, and may be a zoonotic infection from pigs. We present a case of locally acquired hepatitis E infection in a patient with previously undiagnosed cirrhosis, which resulted in subacute liver failure and death. We describe our attempt to trace this infection to a free-range pig farm adjacent to the patient's place of employment. Hepatitis E infection should be considered in the differential diagnosis in patients with decompensated chronic liver disease whatever their age or travel history. When found, the prognosis may be poor.

Prevalence and genetic characterization of hepatitis E virus in paired samples of feces and serum from naturally infected pigs.

This study describes the distribution of Hepatitis E virus (HEV) in a naturally infected swine population and the genetic relatedness of HEV strains on swine farms in Spain. Of fecal and serum samples collected from 131 pigs and manure-ditch samples collected from 17 farms, HEV was detected in 16%, 14%, and 59%, respectively, for an overall prevalence rate of 23%. The maximum prevalence rates for feces and serum were in pigs 5 to 12 wk old. A high prevalence of the virus in feces (18%) was observed in sows. Gene sequencing was performed on 6 strains from feces, serum, and manure ditch: the nucleotide identities varied from 81.5% to 99% when compared with those of other strains of genotype 3 isolated from swine. This is the first study in Europe to show the variation in virus distribution by age in feces and serum in a naturally infected swine population.

New culture media for the isolation of Streptococcus mutans and Lactobacillus in the saliva of head- and neck-irradiated patients.

UNLABELLED: The reduction in salivary flow in patients subjected to head and neck irradiation induces changes in the oral microflora and increases the risk of oral mucosal infections. The frequent presence of fungi, particularly Candida, in the oral environment of these patients complicates identification of the most important cariogenic bacteria with the commercial CRT Bacteria (Ivoclar Vivadent) culture media. Such identification is important for the application of chemical measures to control cariogenic bacteria in these patients, since it has been shown that simple fluoride application is unable to control caries in this population. OBJECTIVE: The aim of this study was to obtain a simple medium that inhibits Candida spp. growth and allows the specific growth of Streptococcus mutans and Lactobacillus spp. Thus, reliable counts of cariogenic species can be achieved. MATERIALS AND METHODS: Stimulated saliva samples from 30 head- and neck-radiotherapy patients were seeded in commercial CRT Bacteria culture medium and in two different media designed by our group: mitis salivarius bacitracin agar (MSBA), containing 5% potassium tellurite and fluconazole 64 microg/ml (MSBTPF) for the isolation of Streptococcus; and Man, Rogosa and Sharpe (MRS) agar, containing bacitracin 0.2 U/ml and fluconazole 32 microg/ml (MRSBF) for the isolation of Lactobacillus spp. RESULTS: Candida growth was inhibited 100% in the media developed in this study. In all the samples seeded, growing of colonies in MRSBF was identified as Lactobacillus, while in CRT Bacteria for Lactobacillus spp. this species was only isolated in 48.1% of the samples. S. mutans was identified in 71.4% of the colonies that grown in MSBTPF medium, while in CRT Bacteria for S. mutans, this species was only identified in 35% of the colonies obtained. CONCLUSION: The culture medium developed in the present study was able to inhibit the 100% of Candida spp. growth. These new media permit reliable counts of cariogenic bacteria in irradiated patients.

Detection of hepatitis E virus shedding in feces of pigs at different stages of production using reverse transcription-polymerase chain reaction.

The aim of this study was to determine at which production stages hepatitis E virus (HEV) is shed by the highest number of pigs and to estimate the relative risk associated with each stage. For this purpose, 146 fecal samples of pigs from 21 farms were studied. In addition, 1 sample from the manure ditch and another sample of drinking water, collected directly from the trough located in the pen, were taken from 16 farms. HEV RNA was detected in fecal samples from 34 pigs (23.29%). The production stages in which most pigs excreted HEV were weaners (41.7%) and pigs in the first month of feeding (60%). The results of the statistical analysis showed that the principal significant risk stage in HEV shedding was the first month of feeding (odds ratio [OR] 19.5, 95% CI 3.59-106.07, P = 0.001) followed by the weaners stage (OR 9.3, 95% CI .78-48.42, P = 0.008). In 8 out of 16 farms tested (50%) HEV RNA was detected in raw manure and in the water trough of only 1. Detection of HEV in manure ditches raises the concern of how to deal with manure of swine origin, because it is used as soil fertilizer.

[Canine onychomycosis produced by Microsporum gypseum. A case report]. / Onicomicosis canina producida por Microsporum gypseum. A propósito de un caso.

One case of severe canine onychomycosis is described. The aetiological agent was identified as Microsporum gypseum. The incidence of this fungus in this kind of pathology is discussed, with special attention to the successful treatment with topic enilconazole and systemic griseofulvin.

Multilocus genotyping of Giardia duodenalis in lambs from Spain reveals a high heterogeneity.

Fecal specimens from 120 lambs in Valencia (Spain) were analyzed for Giardia duodenalis by IFA and nested-PCR using the beta giardin (bg), glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and small subunit ribosomal RNA (ssurRNA) genes. The highest prevalence was obtained using the ssurRNA gene (89.2%), whereas values from other techniques ranged from 64.1% to 69.2%. Sequences of the ssurRNA showed a high proportion of assemblage A or mixed assemblage A/E samples (55.1% and 25.2%, respectively). When the other 3 loci were analyzed, between 6.5% and 15.4% were found to be assemblage A or A/E, respectively. Nested PCR for the tpi gene was the most variable of the targets employed. Twelve new sequences of gdh and tpi for G. duodenalis from sheep were found. Multilocus genotyping resulted in 63 patterns from the 71 samples sequenced at the four loci. This high variability among isolates possibly reflects the high frequency of mixed infections.

Fulminant hepatitis E in a woman taking oral contraceptive medication.

We describe a fulminant autochthonous hepatic failure caused by hepatitis E (HEV) in a patient admitted in our hospital for liver-transplant evaluation. The only risk factor recorded for this severe course was the use of oral contraceptives that are known to mimic a hormonal status similar to pregnancy. The diagnosis was based on the presence of IgG and IgM anti-HEV in the serum of the patient and confirmed by the isolation of a strain of HEV genotype 3f from a blood sample obtained the fourth day after hospital admission. HEV genotype 3 is present in human and swine populations in Spain. The patient began to recover while waiting for a liver transplant. To our knowledge, this is the first report of fulminant hepatitis E in a non-pregnant European patient on oral contraceptives.

Occurrence and genotypes of Giardia isolated from lambs in Spain.

Three hundred and eighty six faecal specimens were randomly collected from 1- to 3-month-old lambs from 16 farms in Spain to investigate the presence of different genotypes of Giardia duodenalis. Individual specimens were examined by IFA (Immunofluorescence assay) and beta-giardin PCR polymerase chain reaction. Cysts of G. duodenalis were shed by lambs in every flock analyzed, showing a prevalence by farms of 100%. The average prevalence of G. duodenalis for the 386 specimens was 42%, ranging from 8.3 to 80% depending on the farm. beta-giardin PCR positive samples were sequenced to determine the genotypes present at each farm and seven new subtypes of beta-giardin Assemblage E are reported in this study. In each farm, one to six different beta-giardin subtypes were found, showing the high variability of the target. Also, one flock had the zoonotic Assemblage A. This is the first report of Giardia subgenotype A-1 in sheep in Spain.

Occupational exposure to hepatitis E virus (HEV) in swine workers.

The aim of this work was to study the prevalence of hepatitis E virus (HEV) and the risk factors for the acquisition of the virus in a population in contact with swine and unexposed to swine. A total of 198 individuals, 97 unexposed (49%) and 101 exposed (51%) to swine, were tested for the presence of HEV infection. The prevalence of anti-HEV IgG in the exposed group was 18.8% versus 4.1% in the unexposed to swine group. People exposed to swine were observed to be 5.4 times (P = 0.03) at risk of having anti-HEV IgG. Ten (52.6%) of the IgG-positive individuals showed two concomitant risk factors: untreated water consumption and exposure to swine. These data support that HEV infection should be treated as a vocational illness in swine workers. Therefore, systematic application of hygiene measures in this collective is highly recommended to avoid the exposition to this virus.

Autochthonous hepatitis E infection in a slaughterhouse worker.

We report the first hepatitis E infection case detected in a slaughterhouse worker. The identified strain belonged to genotype 3, subtype 3f. Partial sequence analysis of the strain isolated from his serum showed a percentage of nucleotide homology ranging from 83.4% up to 97.3% compared with European human and swine strains, respectively. These findings point strongly to hepatitis E virus as a vocationally acquired illness by means of the manipulation of infected organs from pigs.

Diagnostic techniques to detect cryptic leishmaniasis in dogs.

This study of several techniques for detecting cryptic leishmaniasis in dogs from areas in Spain where Leishmania infantum is highly endemic concludes that immunological techniques (enzyme-linked immunosorbent assay, immunofluorescence antibody test, Western blotting, delayed-type hypersensitivity reaction, and in vitro lymphocyte proliferation assay) do not clearly differentiate between noninfected and infected asymptomatic dogs and that culture and PCR are more reliable diagnostic tools.

Practical approach for typing strains of Leishmania infantum by microsatellite analysis.

Currently the universally accepted standard procedure for characterizing and identifying strains of Leishmania is isoenzyme analysis. However, in the Mediterranean area, despite their very wide geographical distribution, most Leishmania infantum strains belong to zymodeme MON-1. In order to increase our understanding of polymorphism in strains of L. infantum, we developed PCR assays amplifying 10 microsatellites and sequenced PCR products. The discriminative power of microsatellite analysis was tested by using a panel of 50 L. infantum strains collected from patients and dogs from Spain, France, and Israel, including 32 strains belonging to zymodeme MON-1, 8 strains belonging to zymodemes MON-24, MON-29, MON-33, MON-34, or MON-80, and 10 untyped strains. Five of the microsatellites were polymorphic, revealing 22 genotypes, whereas the five remaining microsatellites were not variable. In particular, MON-1 strains could be separated into 13 different closely related genotypes. MON-33 and MON-34 strains also gave two additional genotypes closely related to MON-1, while MON-29, MON-24, and MON 80 strains exhibited more divergent genotypes. Among the foci examined, the Catalonian focus displayed a high polymorphism, probably reflecting isoenzyme polymorphism, while the Israeli focus exhibited a low polymorphism that could be consistent with the recent reemergence and rapid spread of canine leishmaniasis in northern and central Israel. The strains originating from the south of France and the Madrid, Spain, area displayed significant microsatellite polymorphism even though they were monomorphic by isoenzyme analysis. In conclusion, microsatellite polymorphism exhibits a high discriminative power and appears to be suitable for characterization of closely related strains of L. infantum in epidemiological studies.