Marsupialization for the treatment of nictitating membrane cyst in a dog: case report / Marsupialização para o tratamento de cisto em membrana nictitante de cão: relato de caso

This study aims to describe the first Brazilian report of a nictitating membrane cyst's surgical treatment in a dog. A 6-month-old female French Bulldog presented at HOSVET-UNIME with a reddish mass-like structure in the medial canthus of both eyes, with a history of recurrent third eyelid gland prolapse previously treated with two surgeries performed at another clinic. Physical examination revealed a third eyelid gland prolapse in the right eye and a cyst in the left eye's third eyelid. The animal was submitted to surgical correction of the right eye's third eyelid prolapse using pocket technique and of the left eye's third eyelid using marsupialization technique for the cyst's treatment. 180 days after th1e surgical procedure no recurrence was observed. The marsupialization technique for the treatment of a third eyelid's lacrimal cyst in a dog allowed the maintenance of its gland and prevented the formation of a new cystic cavity.(AU)

O objetivo do presente trabalho é descrever o primeiro relato no Brasil de tratamento cirúrgico de um cisto da membrana nictitante em um cão. Um Buldogue Francês, fêmea, seis meses, foi atendido no Hosvet-Unime, com queixa de aumento de volume avermelhado no canto medial de ambos os olhos, com histórico de recidiva de prolapso de glândula da terceira pálpebra, onde haviam sido realizadas duas cirurgias anteriormente em outro local. Ao exame físico, foi observado prolapso de glândula da terceira pálpebra no olho direito e a presença de um cisto na terceira pálpebra do olho esquerdo. O animal foi submetido ao procedimento cirúrgico de sepultamento de glândula da terceira pálpebra no olho direito e uma marsupialização na terceira pálpebra do olho esquerdo para o tratamento do cisto. Cento e oitenta dias após o procedimento cirúrgico, não foi observada recidiva. A técnica de marsupialização para tratamento de cisto lacrimal na terceira pálpebra em um cão possibilitou a manutenção da sua glândula e impediu a formação de nova cavidade cística.(AU)

Molecular identification of Ehrlichia sp. in a beef cattle from Brazilian Cerrado - case report / Identificação molecular de Ehrlichia sp em um bovino do Cerrado brasileiro - relato de caso

Ehrlichia infections in cattle are frequent in Africa but have also been reported in Brazil and North America. This paper reports natural infection by Ehrlichia sp. associated with Babesia bigemina and Anaplasma marginale in a calf in the municipality of Campo Grande, state of Mato Grosso do Sul, Brazil, presenting polioencephalomalacia. The molecular evidence, based on a fragment of the dsb gene, indicates a species of Ehrlichia genetically related to Ehrlichia canis and other species of the genus found in the tick Rhipicephalus (Boophilus) microplus and a calf from Brazil (99 to 100% identity). It was not possible to associate the clinical signs with Ehrlichia infection due to co-infections and histological evidence of another disease. However, the circulation of the bacteria in bovines in Brazilian Cerrado was confirmed and more attention should be given to clinical suspicion of tick-borne pathogens in cattle to clarify the pathogenic potential of Ehrlichia sp.(AU)

Infecções por Ehrlichia em bovinos são frequentes na África, mas também foram relatadas no Brasil e na América do Norte. Este artigo relata uma infecção natural por Ehrlichia sp. associado a Babesia bigemina e Anaplasma marginale em um bezerro, no município de Campo Grande, Mato Grosso do Sul, Brasil, o qual apresentava polioencefalomalácia. A evidência molecular, baseada em um fragmento do gene dsb, indica uma espécie de Ehrlichia geneticamente relacionada a Ehrlichia canis e outras espécies do gênero encontradas no carrapato Rhipicephalus (Boophilus) microplus e em um bezerro do Brasil (99 a 100% de identidade). Não foi possível associar os sinais clínicos à infecção por Ehrlichia devido a coinfecções e evidências histológicas de outra doença. No entanto, a circulação da bactéria em bovinos no Cerrado brasileiro foi confirmada, e mais atenção deve ser dada à suspeita clínica de patógenos transmitidos por carrapatos em bovinos para esclarecer o potencial patogênico de Ehrlichia sp.(AU)

Identification of SNP c. -22G>A in the melanophilin gene from a dog with color dilution alopecia: case report / Identificação do SNP c. -22G>A no gene da melanophilina em um cão com alopecia por diluição da cor: relato de caso

Mutant color alopecia is an ectodermical defection of color dilution, characterized by partial alopecia, dry, shine-less hair, and peeling and papule. Melanization damages also occur on the cortical structure of the affected hair. The animals affected have big melanin grains with irregular shape on the basal keratinocytes, also on the hair matrix cells and rod. Therefore, there is not a specific treatment that makes any difference on the syndrome evolution. Although in some animals, it is possible to use weekly showers with benzyl peroxide to reduce seborrhea formation and secondary infections. There is evidence that the condition in dogs is caused by a single nucleotide polymorphism in the gene encoding the melanophilin protein. In the present study the identification of the SNP c.-22G>A in the melanophilin gene of a Dachshund breed dog with clinical and histopathologic evidence of color dilution alopecia is reported.(AU)

Alopecia por diluição da cor é um defeito ectodérmico caracterizado por alopecia parcial, pelagem seca e sem brilho, escamação e pápulas em áreas com defeitos na melanização e na estrutura cortical dos pelos. Os animais acometidos têm grânulos de melanina grandes e com formato irregular nos ceratinócitos basais, nas células da matriz dos pelos e nas hastes pilosas. Não existe tratamento específico que altere a evolução da síndrome, mas, em alguns animais, podem ser benéficos banhos semanais com xampu de peróxido de benzoíla, para reduzir a formação de seborreia e infecções secundárias. Há evidências de que a condição em cães é causada por uma mutação de ponto no gene que codifica a proteína melanophilina. No presente estudo, é relatada a identificação da mutação SNP c.-22G>A no gene da melanophilina em um cão da raça Dachshund com evidências clínicas e histopatológicas de alopecia por diluição da cor.(AU)

Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS: We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS: Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS: Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Immune function surveillance: association with rejection, infection and cardiac allograft vasculopathy.

BACKGROUND: Rejection, cardiac allograft vasculopathy (CAV), and infection are significant causes of mortality in heart transplantation recipients. Assessing the immune status of a particular patient remains challenging. Although endomyocardial biopsy (EMB) and angiography are effective for the identification of rejection and CAV, respectively, these are expensive, invasive, and may have numerous complications. The aim of this study was to evaluate the immune function and assess its utility in predicting rejection, CAV, and infection in heart transplantation recipients. METHODS: We prospectively obtained samples at the time of routine EMB and when clinically indicated for measurement of the ImmuKnow assay (IM), 12 cytokines and soluble CD30 (sCD30). EMB specimens were evaluated for acute cellular rejection, and antibody-mediated rejection (AMR). CAV was diagnosed by the development of angiographic coronary artery disease. Infectious episodes occurring during the next 30 days after testing were identified by the presence of positive bacterial or fungal cultures and/or viremia that prompted treatment with antimicrobials. RESULTS: We collected 162 samples from 56 cardiac transplant recipients. There were 31 infection episodes, 7 AMR, and 4 CAV cases. The average IM value was significantly lower during infection, (P = .04). Soluble CD30 concentrations showed significantly positive correlation with infection episodes, (P = .001). Significant positive correlation was observed between interleukin-5(IL-5) and AMR episodes (P = .008). Tumor necrosis factor-α and IL-8 showed significant positive correlation with CAV (P = .001). CONCLUSIONS: Immune function monitoring appears promising in predicting rejection, CAV, and infection in cardiac transplantation recipients. This approach may help in more individualized immunosuppression and it may also minimize unnecessary EMBs and cardiac angiographies.

A comparative study of the histopathology and immunohistochemistry of pythiosis in horses, dogs and cattle.

Twenty-one cases of pythiosis in horses (n = 10), dogs (n = 9) and cattle (n = 2) were investigated. The aetiology in all cases was confirmed by immunohistochemistry. Data related to the clinical course and outcome and localization of the lesions were obtained from pathology reports. The equine lesions consisted of fibrotic tissue with multiple, often coalescing, areas of immature granulation tissue encircling eosinophilic cores. Affected dogs had gastrointestinal and/or cutaneous lesions with either or both of a granulomatous/pyogranulomatous or necrotizing eosinophilic inflammatory reaction. In cattle, cutaneous lesions were characterized by multifocal to coalescing granulomas with surrounding fibrosis. The number of intralesional hyphae, the distribution of hyphae, the presence of angioinvasion and the nature of the local inflammatory reactions were associated with the different types of lesions observed.

Morphological factors as indicators of malignancy of squamous cell carcinomas in cattle exposed naturally to bracken fern (Pteridium aquilinum).

Squamous cell carcinomas (SCCs) of the upper digestive tract (UDT) of cattle have been associated with chronic bracken fern (Pteridium aquilinum) toxicity and infection with bovine papillomavirus type-4. These tumours share some morphological similarities with human head and neck SCCs. In this study, morphological changes were correlated with the biological behaviour of 40 alimentary SCCs in cattle grazing on pastures with high bracken content. The majority of SCCs were localized to the cranial and caudal regions of the UDT (almost 45% each). More than 60% of the tumours were well differentiated and were found mostly in the cranial region. Metastasis occurred in 58% of the cases, mostly to regional lymph nodes. All poorly differentiated SCCs had evidence of metastasis. Morphological patterns characterized by islands and ribbons of neoplastic keratinocytes were more prominent in well differentiated SCCs. These patterns varied greatly in moderately differentiated SCCs. Poorly differentiated tumours were characterized by the presence of cellular aggregates and individual cells and these tumours had more marked desmoplasia. A significant positive association was established between lymphoplasmacytic inflammatory infiltration and tumour-associated tissue eosinophilia. Evaluation of argyrophylic nucleolar organizer regions (AgNORs) revealed higher proliferation indices in poorly differentiated tumours than in moderately or well differentiated lesions. There was significant correlation between the AgNOR index and histological grading. The morphological factors analyzed were all related to histological grading, which is the major factor predicting the biological behaviour of SCCs in cattle naturally exposed to bracken fern.

Immunoglobulin (Ig) M antibody against myelin associated glycoprotein (MAG): A comparison of methods.

The presence of immunoglobulin (Ig)M antibody against myelin associated glycoprotein (MAG) has been associated with autoimmune demyelinating, sensorimotor neuropathies. Approximately 50% of patients with IgM paraproteinemia and associated peripheral neuropathy possess antibodies against MAG. These autoantibodies are thought to interfere with the process of myelination, myelin maintenance, or axon-Schwann cell interaction. The detection of these autoantibodies is useful to the clinician and is suggestive of active demyelination in a peripheral neuropathy. Our objective in this study was to compare the results obtained using three different methods (dual enzyme immunoassay [EIA], immunofluorescent antibody [IFA] and Western blot [WB]) for detecting IgM antibody against MAG in patients suspected of having autoimmune demyelinating neuropathies. Since the dual EIA utilized two different antigens, results from this assay were separated into two groups: MAG and sulfate-3-glucuronyl paragloboside (SGPG). When compared to WB (gold standard), percent agreement, sensitivity, and specificity for EIA and IFA are as follows: MAG EIA (68.3, 100.0, and 60.6); SGPG EIA (95.1, 100.0, and 93.9); and myelin IFA (97.6, 100.0, and 97.0). The authors conclude that the SGPG EIA and myelin IFA compared well with the standard WB method when detecting IgM antibody against MAG (100 kD). Many sera demonstrated reactivity on the MAG EIA that were negative by WB (100 kD glycoprotein). The authors recommend screening for MAG IgM in suspected patient sera by SGPG EIA or myelin IFA and utilizing these same methods to titer sera confirmed positive by WB.

IgA antibodies against endomysium and transglutaminase: a comparison of methods.

Recently, the endomysial antigen has been identified as the protein cross-linking enzyme known as tissue transglutaminase (tTG). Our objective was to compare a novel enzyme immunoassay (EIA) that detects IgA antibody against tTG to two standard IFA methods utilizing thin tissue sections of rat kidney/rat stomach (KS) and distal primate esophagus (PE) as substrates to detect IgA antibody against endomysium (EMA). Sera from 100 patients suspected of having gluten-sensitive enteropathy (GSE) and 23 sera possessing various antibodies used for EIA cross-reactivity studies were included. Additional tests, performed routinely in our laboratory, were utilized to further assess sera from patients suspected having GSE. These tests include anti-gliadin IgA antibody (AGA) and anti-reticulin IgA antibody (ARA) and are part of the European Society for Pediatric Gastroenterology and Nutrition (ESPGAN) revised criteria for diagnosing GSE. When compared to IFA using KS, the tTG EIA had a sensitivity of 87.5%, was 97.1% specific, and had an overall agreement of 94.0%. When compared to IFA using PE, the tTG EIA had a sensitivity of 92.6%, was 93.2% specific, and had an overall agreement of 93.0%. When the KS IFA was compared to the PE IFA for EMA, the KS IFA had a sensitivity of 96.3%, was 91.8% specific, and had an overall agreement of 93.0%. The majority of sera that were positive for tTG but were negative by IFA (KS, n = 2/PE, n = 5) possessed IgA antibodies against gliadin and/or reticulin. Five of six sera with negative results by PE IFA were positive by the KS IFA and possessed one or more antibodies to tTG and/or gliadin and/or reticulin. We conclude that the tTG EIA compares well to both KS and PE IFAs when detecting IgA antibody against endomysium. We do not recommend the use of PE to detect EMA primarily because of the inconsistencies (i.e., tissue selection, quality, and preparation) and limited availability of commercially prepared PE tissue.

Comparison of four enzyme immunoassays with a western blot assay for the determination of type-specific antibodies to herpes simplex virus.

Most current enzyme immunoassays (EIAs) differentiate inadequately between types 1 and 2 herpes simplex virus (HSV) antibodies since significant cross-reactivity exists. We compared 4 IgG type-specific EIAs using a Western blot assay for resolution of discrepant results. The Diamedix had sensitivities of 100% for types 1 and 2 but specificities of only 71% and 61%, respectively. The cross-reactivity rate was 82% in positive samples tested. For HSV types 1 and 2, the Zeus sensitivities were 92% and 98%, respectively; specificities were 72% and 79%, respectively; the cross-reactivity rate was 54%. For HSV types 1 and 2, the Wampole sensitivities were 98% and 95%, respectively; specificities were 68% and 85%, respectively; the cross-reactivity rate was 47%. For HSV types 1 and 2, the Meridian sensitivities were 98% and 90%, respectively; specificities were 96% and 100%, respectively; no cross-reactivity was found between positive samples tested. While the Diamedix, Zeus, and Wampole assays showed good sensitivity, they lacked type specificity. The Meridian EIA offers the highest specificity along with no observed cross-reactivity. This EIA may be an easier, reliable alternative to Western blot for the determination of HSV type-specific antibodies.

Evaluation of a PCR probe capture assay for the detection of Toxoplasma gondii. Incorporation of uracil N-glycosylase for contamination control.

Toxoplasma gondii is a cyst-forming parasite of clinical relevance in humans primarily because of the neurologic abnormalities it can cause. In some clinical circumstances, it is desirable to detect the pathogen directly. We modified a commercially available Toxoplasma polymerase chain reaction (PCR) probe capture assay by incorporating uracil N-glycosylase (UNG) to prevent carryover amplicon contamination. In addition, UNG inactivation and DNA denaturation were accomplished chemically to simplify the DNA hybridization to the capture probe. The incorporation of UNG effectively eliminated carryover contamination; the probe capture assay showed a log increase in detection sensitivity compared with standard agarose gel electrophoresis. To assess sensitivity and possible inhibition of amplification, different sample types were spiked with Toxoplasma organisms. After DNA extraction and PCR amplification, a sensitivity of 2 tachyzoites for the assay was determined in buffered saline, cerebrospinal fluid (CSF), serum, and amniotic fluid; 20 tachyzoites for whole blood; and 200 tachyzoites for brain tissue. An additional 20 human serum and CSF samples submitted for Toxoplasma serologic testing were run by the PCR method. Of these, only an IgM-positive CSF sample was PCR positive. The Toxoplasma PCR probe capture assay showed good sensitivity and was not substantially inhibited by several different clinically relevant samples.

Comparison of three different methods for measuring classical pathway complement activity.

The complement system plays an important role in host defense against infection and in most inflammatory processes. The standard 50% hemolytic complement (CH50) assay is the most commonly used method of screening patient sera for functional activity of the classical complement pathway. Our objective in this study was to compare two newer methods (the enzyme immunoassay and the liposome immunoassay) to a commercial CH50 assay for measuring total classical complement activity. We conclude that both newer methods compare well with a CH50 assay and are equally sensitive in screening routine clinical sera.

Immunoglobulin A antibodies to Helicobacter pylori.

Serological testing for immunoglobulin G (IgG) antibodies to Helicobacter pylori has proven useful in supporting the diagnosis of infection with this organism, but the clinical value of IgA antibodies in H. pylori-related gastritis remains controversial. The purpose of our study was to determine the frequency of IgA-positive IgG-negative patients with symptoms of gastrointestinal (GI) disorders, thus assessing the clinical utility of IgA testing for H. pylori-related gastritis. It was found previously that the frequency of infected individuals in this category (IgA positive and IgG negative) is about 2%, but a large number of IgG-negative patients with GI disorders suggestive of H. pylori infection have not been investigated until now.

Immunologic response to Bartonella henselae as determined by enzyme immunoassay and Western blot analysis.

Bartonella henselae is now regarded as the etiologic agent of cat-scratch disease and a cause of bacillary angiomatosis. We examined the human immune response to Bartonella henselae infection using a newly developed enzyme immunoassay (EIA) and a Western blot procedure using outer-membrane proteins. The EIA showed 98.6% and 91.4% agreement with an indirect fluorescence method (IFA) for detection of IgM and IgG antibodies, respectively. By using Western blot analysis, reactivity to an 8-kd band showed significant correlation with positive results by the IgM IFA and EIA. In contrast, reactivity to 209-, 208.5-, 208-, 116-, and 80-kd bands was identified only in positive IgG IFA serum samples. The EIA and Western blot should be useful tests in determining the antibody response to B. henselae infection and may also be important in determining the critical epitopes in the host-parasite interaction of this organism.

Detection of Epstein-Barr viral DNA in serum using rapid-cycle PCR.

Our study describes the comparison of a rapid nested PCR assay to standard serology techniques for the detection of Epstein-Barr virus (EBV) in serum. The sera of 81 patients with suspected EBV infection were analyzed; 54 were positive for one or more of the standard serology markers, i.e., IgM viral capsid antigen (VCA), IgG-VCA, Epstein-Barr nuclear antigen 1 (EBNA-1), and early antigen (EA), and 27 were negative for all serology markers. The sera from 15 normal healthy blood donors were also included. No EBV DNA was detected in any of the 15 blood donor samples or in any of the 27 samples with negative serology results. Eleven samples (20%) of the 54 with positive EBV serology results were positive for EBV DNA. Of these samples, 9 were EBV IgM-VCA positive and anti-EBNA negative, suggesting acute infection. One of the 11 samples had high titers of IgM-VCA, IgG-VCA, anti-EBNA, and anti-EA. The last of the 11 samples was from a patient with acute infectious mononucleosis without sufficient sample volume for EBV serology testing. Seventeen of the total 96 samples from the study were IgM-VCA positive and anti-EBNA negative and 9 of these 17 samples (53%) tested positive for EBV DNA. These data suggest that the detection of EBV DNA by PCR in serum may be a useful indicator of active infection rather than latent virus.

Screening for antinuclear antibodies by enzyme immunoassay.

Indirect fluorescent antibody (IFA) ia most widely used method in clin clinical laboratories to screen for autoantibodies against a wide variety of nuclear antigens. Recently, a number of antinuclear antibody (ANA) enzyme immunoassay (EIA) screens have become commercially available and claim to be an alternative method to screen for ANAs. Given the subjectivity of technical interpretation of IFA and the high number of ANA negative samples, a suitable EIA method for ANA screening would be beneficial to clinical laboratories with large samples volumes. Five ANA EIA screens were compared (Elias, Helix, Sanofi, TheraTest and Zeus) to IFA using a human epithelial cell line (HEp-2). Sera from 601 patients submitted to our reference laboratory for autoimmune testing, and from 202 normal healthy blood donors, were included in this study. Samples with discordant results between IFA and EIA were further analyzed using single antigen EIAs for SSA, SSB, Sm, RNP, Scl-70, histones, dsDNA, and ssDNA. Analyses were based on clinically significant IFA titers of > or equal to 1:160 as positive and <1:40 as negative. When compared to IFA, agreement, sensitivity and specificity for each ANA EIA screen were as follows: Elias: 87.0%, 69.5% and 97.9%; Helix: 94.6%, 90.2%, and 97.3%; Sanofi: 95.0%, 93.7%, and 95.9%; TheraTest: 95.3%, 97.7%, and 93.5%; Zeus: 87.1%, 96.2%, and 81.4%, respectively. In conclusion, screening for ANAs by EIA using several commercial assays was both sensitive and specific when compared to IFA. Moreover, the EIA is objective and much less labor intensive when screening a large number of clinical specimens. None of the EIAs were 100% sensitive and, thus, may fail to detect a few of the nonspecific ANAs that demonstrate atypical as well as classical IFA patterns. The advantages of employing these nonsubjective assays to screen out the vast majority of ANA negative sera is clear. The authors still recommend confirming titers and patterns of sera with positive EIA screens using classical IFA methods employing HEp-2 cells.

Comparison of three commercially available enzyme immunoassays for the screening of autoantibodies to extractable nuclear antigens.

The initial screening test used in the diagnosis of connective tissue diseases is based on the detection of antinuclear antibodies (ANA) by indirect immunofluorescence (IFA). When the ANA screen is positive, it is often useful to determine the specificity of the autoantibody to a series of extractable nuclear antigens (ENA), a procedure that has been classically performed by double immunodiffusion. Testing large numbers of clinical specimens for autoantibodies to ENA by double diffusion techniques can be time-consuming and expensive. ENA screening systems that employ enzyme immunoassay (EIA) technology have recently become commercially available. We compared three EIA ENA assays to classic Ouchterlony double diffusion techniques. Furthermore, the sensitivity of each antigen and methodology (including ANA immunofluorescence using HEp-2 cells) was tested using ENA positive sera possessing single autoantibodies. Two of the three EIAs that detected immunoglobulin G type autoantibodies to Smith (Sm), ribonucleoprotein (RNP), Sjögren's syndrome-associated antigens Ro (SSA) and La (SSB), were provided by INOVA and Sigma Diagnostics. A third EIA, which also included scleroderma-associated antigen 70 (SCL-70/DNA-topoisomerase I) and histidyl-tRNA synthetase (Jo-1) in addition to the four previously stated antigens, was provided by Clark Laboratories. This latter ENA screen detected IgG, IgA, and IgM type autoantibodies. Included in the study were sera covering a wide variety of anti-nuclear and other autoantibodies. Sensitivity was 100% for all EIA ENA screens when compared to Ouchterlony double diffusion and specificity exceeded 95% in each case. Sensitivity studies showed Ouchterlony to be less sensitive than EIA when detecting low levels of autoantibodies to ENA.(ABSTRACT TRUNCATED AT 250 WORDS)