Using a top predator as a sentinel for environmental contamination with pathogenic bacteria: the Iberian wolf and leptospires

The Iberian wolf (Canis lupus) is the top predator in the Iberian environments in which it lives, feeding on a wide range of species, thus encountering a wide range of disease agents. Therefore, the wolf can serve as sentinel of environmental contamination with pathogens. We investigated the exposure of free-living wolves to 14 serovars of Leptospira interrogans sensu lato. Kidney samples from 49 wolves collected from 2010-2013 in northwestern Spain were analysed by culture, direct immunofluorescence and polymerase chain reaction. Tissue fluids were analysed for antibodies by a microscopic agglutination test. Ten wolves (observed prevalence: 20%, 95% confidence interval = 11-33%) showed evidence of contact with leptospires, eight through direct detection and nine through serology (7 wolves were positive according to both techniques). Titres below the cut-off level were also detected in seven cases. Serovars confirmed were Canicola (n = 4), Icterohaemorrhagiae (n = 3) and Sejroë, Ballum and Grippotyphosa (n = 1 each), indicating that wolves were infected with serovars for which dogs, rodents and ungulates, are the natural hosts and supporting the utility of the wolf and other large predators as environmental sentinels for pathogens.

Comparison of three diagnostic techniques for the detection of leptospires in the kidneys of wild house mice (Mus musculus)

Forty-one wild house mice (Mus musculus) were trapped in an urban area, near railways, in Santa Fe city, Argentina. Both kidneys from each mouse were removed for bacteriological and histological examination. One kidney was inoculated into Fletcher semi-solid medium and isolates were serologically typed. The other kidney was microscopically examined after hematoxylin-eosin, silver impregnation and immunohistochemical stains. Leptospires, all of them belonging to the Ballum serogroup, were isolated from 16 (39%) out of 41 samples. The presence of the agent was recorded in 18 (44%) and in 19 (46%) out of 41 silver impregnated and immunohistochemically stained samples respectively. Additionally, leptospires were detected in high number on the apical surface of epithelial cells and in the lumen of medullary tubules and they were less frequently seen on the apical surface of epithelial cells or in the lumen of the cortical tubules, which represents an unusual finding in carrier animals. Microscopic lesions consisting of focal mononuclear interstitial nephritis, glomerular shrinkage and desquamation of tubular epithelial cells were observed in 13 of 19 infected and in 10 of 22 non-infected mice; differences in presence of lesions between infected and non-infected animals were not statistically significant (P=0,14). The three techniques, culture, silver impregnation and immunohistochemistry, had a high agreement (k³0.85) and no significant differences between them were detected (P>0.05). In addition, an unusual location of leptospires in kidneys of carrier animals was reported, but a relationship between lesions and presence of leptospires could not be established.

Molecular confirmation of ovine herpesvirus 2-induced malignant catarrhal fever lesions in cattle from Rio Grande do Norte, Brazil

Molecular findings that confirmed the participation of ovine herpesvirus 2 (OVH-2) in the lesions that were consistent with those observed in malignant catarrhal fever of cattle are described. Three mixed-breed cattle from Rio Grande do Norte state demonstrated clinical manifestations that included mucopurulent nasal discharge, corneal opacity and motor incoordination. Routine necropsy examination demonstrated ulcerations and hemorrhage of the oral cavity, corneal opacity, and lymph node enlargement. Significant histopathological findings included widespread necrotizing vasculitis, non-suppurative meningoencephalitis, lymphocytic interstitial nephritis and hepatitis, and thrombosis. PCR assay performed on DNA extracted from kidney and mesenteric lymph node of one animal amplified a product of 423 base pairs corresponding to a target sequence within the ovine herpesvirus 2 (OVH-2) tegument protein gene. Direct sequencing of the PCR products, from extracted DNA of the kidney and mesenteric lymph node of one cow, amplified the partial nucleotide sequences (423 base pairs) of OVH-2 tegument protein gene. Blast analysis confirmed that these sequences have 98-100% identity with similar OVH-2 sequences deposited in GenBank. Phylogenetic analyses, based on the deduced amino acid sequences, demonstrated that the strain of OVH-2 circulating in ruminants from the Brazilian states of Rio Grande do Norte and Minas Gerais are similar to that identified in other geographical locations. These findings confirmed the active participation of OVH-2 in the classical manifestations of sheep associated malignant catarrhal fever.

Outbreaks of canid herpesvirus 1 disease in puppies in southern Brazil

Abstract: Canid herpesvirus 1 (CHV-1) is a widespread pathogen of dogs and produces infertility, abortions and severe systemic disease in young puppies. Clinical data indicate the circulation of CHV-1 among Brazilian dogs yet definitive diagnosis has rarely been accomplished. This article describes the clinicopathological findings of four independent cases/outbreaks of neonatal disease by CHV-1 in Bulldog puppies followed by virus identification and genetic characterization. Three events occurred in a kennel holding dogs of different breeds at reproductive age (March 2013, October 2013 and April 2014). Puppies from three French or English Bulldog litters, aging 9 to 30 days were affected, presenting dyspnea, agonic breathing, pale mucous, abdominal pain and tension, evolving to death within about 24 hours. At necropsy, the puppies presented necrohemorrhagic hepatitis, multifocal and moderate necrohemorrhagic nephritis and fibrinonecrotic interstitial pneumonia. Virus isolation was positive in clinical specimens from one litter and CHV-1 DNA was detected by PCR in tissues from all four cases. Virus-neutralizing assays with samples of the affected kennel revealed 9/12 adult animals with high antibody titers to CHV-1. Nucleotide sequencing of glycoprotein B, C and D genes revealed 99-100% of identity among the viruses and with CHV-1 sequences available in GenBank. Phylogenetic analyses of gC sequences showed a segregation of the samples, even among three isolates from the same kennel. These findings support CHV-1 infection as the cause of disease and death in these dog litters, reinforcing the need for correct etiologic diagnosis, prevention and immunization against CHV-1 in dogs from Southern Brazil.

Origin and pathogenesis of antiphospholipid antibodies

Antiphospholipid antibodies (aPL) are a heterogeneous group of antibodies that are detected in the serum of patients with a variety of conditions, including autoimmune (systemic lupus erythematosus), infectious (syphilis, AIDS) and lymphoproliferative disorders (paraproteinemia, myeloma, lymphocytic leukemias). Thrombosis, thrombocytopenia, recurrent fetal loss and other clinical complications are currently associated with a subgroup of aPL designating the antiphospholipid syndrome. In contrast, aPL from patients with infectious disorders are not associated with any clinical manifestation. These findings led to increased interest in the origin and pathogenesis of aPL. Here we present the clinical features of the antiphospholipid syndrome and review the origin of aPL, the characteristics of experimentally induced aPL and their historical background. Within this context, we discuss the most probable pathogenic mechanisms induced by these antibodies.

Increased expression of p38 mitogen- activated protein kinase is related to the acute renal lesions induced by gentamicin

Mitogen-activated protein kinases (MAPK) may be involved in the pathogenesis of acute renal failure. This study investigated the expression of p-p38 MAPK and nuclear factor kappa B (NF-kappaB) in the renal cortex of rats treated with gentamicin. Twenty rats were injected with gentamicin, 40 mg/kg, im, twice a day for 9 days, 20 with gentamicin + pyrrolidine dithiocarbamate (PDTC, an NF-kappaB inhibitor), 14 with 0.15 M NaCl, im, twice a day for 9 days, and 14 with 0.15 M NaCl , im, twice a day for 9 days and PDTC, 50 mg kg-1 day-1, ip, twice a day for 15 days. The animals were killed 5 and 30 days after the last of the injections and the kidneys were removed for histological, immunohistochemical and Western blot analysis and for nitrate determination. The results of the immunohistochemical study were evaluated by counting the p-p38 MAPK-positive cells per area of renal cortex measuring 0.05 mm². Creatinine was measured by the Jaffé method in blood samples collected 5 and 30 days after the end of the treatments. Gentamicin-treated rats presented a transitory increase in plasma creatinine levels. In addition, animals killed 5 days after the end of gentamicin treatment presented acute tubular necrosis and increased nitrate levels in the renal cortex. Increased expression of p-p38 MAPK and NF-kappaB was also observed in the kidneys from these animals. The animals killed 30 days after gentamicin treatment showed residual areas of interstitial fibrosis in the renal cortex, although the expression of p-p38 MAPK in their kidneys did not differ from control. Treatment with PDTC reduced the functional and structural changes induced by gentamicin as well as the expression of p-p38 MAPK and NF-kappaB. The increased expression of p-p38 MAPK and NF-kappaB observed in these rats suggests that these signaling molecules may be involved in the pathogenesis of tubulointerstitial nephritis induced by gentamicin.

The use of stem cells for the treatment of autoimmune diseases

Autoimmune diseases constitute a heterogeneous group of conditions commonly treated with anti-inflammatory, immunosuppressant and immunomodulating drugs, with satisfactory results in most cases. Nevertheless, some patients become resistant to conventional therapy. The use of high doses of drugs in such cases results in the need for bone marrow reconstitution, a situation which has stimulated research into the use of hematopoietic stem cells in autoimmune disease therapy. Stem cell transplantation in such diseases aims to destroy the self-reacting immune cells and produce a new functional immune system, as well as substitute cells for tissue damaged in the course of the disease. Significant results, such as the reestablishment of tolerance and a decrease in the recurrence of autoimmune disease, have been reported following stem cell transplantation in patients with autoimmune disease in Brazil and throughout the world. These results suggest that stem cell transplantation has the potential to become an important therapeutic approach to the treatment of various autoimmune diseases including rheumatoid arthritis, juvenile idiopathic arthritis, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, Crohn's disease, autoimmune blood cytopenias, and type I diabetes mellitus.

Multiple facets of HIV-associated renal disease

HIV infection has a broad spectrum of renal manifestations. This study examined the clinical and histological manifestations of HIV-associated renal disease, and predictors of renal outcomes. Sixty-one (64% male, mean age 45 years) HIV patients were retrospectively evaluated. Clinical presentation and renal histopathology were assessed, as well as CD4 T-cell count and viral load. The predictive value of histological lesion, baseline CD4 cell count and viral load for end-stage renal disease (ESRD) or death were determined using the Cox regression model. The outcomes of chronic kidney disease (CKD) and ESRD or death were evaluated by baseline CD4 cell count. The percent distribution at initial clinical presentation was non-nephrotic proteinuria (54%), acute kidney injury (28%), nephrotic syndrome (23%), and chronic kidney disease (22%). Focal segmental glomerulosclerosis (28%), mainly the collapsing form (HIVAN), acute interstitial nephritis (AIN) (26%), and immune complex-mediated glomerulonephritis (ICGN) (25%) were the predominant renal histology. Baseline CD4 cell count ≥200 cells/mm3 was a protective factor against CKD (hazard ratio=0.997; 95%CI=0.994-0.999; P=0.012). At last follow-up, 64% of patients with baseline CD4 ≥200 cells/mm3 had eGFR >60 mL·min-1·(1.73 m2)-1 compared to the other 35% of patients who presented with CD4 <200 cells/mm3 (log rank=9.043, P=0.003). In conclusion, the main histological lesion of HIV-associated renal disease was HIVAN, followed by AIN and ICGN. These findings reinforce the need to biopsy HIV patients with kidney impairment and/or proteinuria. Baseline CD4 cell count ≥200 cells/mm3 was associated with better renal function after 2 years of follow-up.

Cocaine/levamisole-induced systemic vasculitis with retiform purpura and pauci-immune glomerulonephritis

Levamisole has been increasingly used as an adulterant of cocaine in recent years, emerging as a public health challenge worldwide. Levamisole-associated toxicity manifests clinically as a systemic vasculitis, consisting of cutaneous, hematological, and renal lesions, among others. Purpura retiform, cutaneous necrosis, intravascular thrombosis, neutropenia, and less commonly crescentic nephritis have been described in association with anti-neutrophil cytoplasmic antibodies (ANCAs) and other autoantibodies. Here we report the case of a 49-year-old male who was a chronic cocaine user, and who presented spontaneous weight loss, arthralgia, and 3 weeks before admission purpuric skin lesions in the earlobes and in the anterior thighs. His laboratory tests on admission showed serum creatinine of 4.56 mg/dL, white blood count 3,800/μL, hemoglobin 7.3 g/dL, urinalysis with 51 white blood cells/μL and 960 red blood cells/μL, and urine protein-to-creatinine ratio 1.20. Serum ANCA testing was positive (>1:320), as well as serum anti-myeloperoxidase and anti-proteinase 3 antibodies. Urine toxicology screen was positive for cocaine and levamisole, with 62.8% of cocaine, 32.2% of levamisole, and 5% of an unidentified substance. Skin and renal biopsies were diagnostic for leukocytoclastic vasculitis and pauci-immune crescentic glomerulonephritis, respectively. The patient showed a good clinical response to cocaine abstinence, and use of corticosteroids and intravenous cyclophosphamide. Last serum creatinine was 1.97 mg/dL, white blood cell count 7,420/μL, and hemoglobin level 10.8 g/dL. In levamisole-induced systemic vasculitis, the early institution of cocaine abstinence, concomitant with the use of immunosuppressive drugs in severe cases, may prevent permanent end organ damage and associate with better clinical outcomes.

Insuficiência renal aguda em pacientes infectados pelo H1N1: correlação clínico-histológica em uma série de casos

INTRODUÇÃO: O vírus Influenza A (H1N1) foi primeiramente descrito em abril de 2009 e, desde então, diversos estudos relataram as características pertinentes à apresentação clínica e ao acometimento pulmonar da doença. Contudo, informações precisas referentes à insuficiência renal aguda (IRA) e às alterações histopatológicas renais nesses pacientes ainda são escassas. OBJETIVO: O objetivo deste estudo é descrever os achados histopatológicos renais de seis pacientes comprovadamente infectados pelo H1N1, que desenvolveram IRA e realizaram biópsia renal, correlacionando-os com os aspectos clínicos. MÉTODOS: Avaliamos seis pacientes do Hospital de Clínicas da UFPR com diagnóstico de H1N1 por PCR viral em 2009 que evoluíram com IRA e que foram submetidos à biópsia renal. Foram revisados os seus prontuários e das lâminas da biópsia renal. RESULTADOS: Todos os casos estudados apresentaram dados clínicos e/ou laboratoriais de IRA, sendo que somente um não apresentou oligúria. À biópsia renal, dois pacientes apresentaram alterações glomerulares: um deles, portador de lupus eritematoso sistêmico, apresentou lesões compatíveis com nefrite lúpica classe III A-C da ISN/RPS 2003 e microangiopatia trombótica focal; outro paciente apresentou glomerulosclerose nodular intercapilar, porém, sem comemorativos clínicos ou laboratoriais de diabetes. Todos os pacientes mostraram graus variáveis de alterações degenerativas vacuolares dos túbulos, com focos de oxalose em dois casos. Dois pacientes possuíam arteriosclerose em grau discreto a moderado. CONCLUSÃO: Em nosso estudo, todos os pacientes apresentarem graus variáveis de alteração degenerativa vacuolar, contudo, não foram encontrados sinais evidentes de necrose tubular aguda, parecendo existir um componente pré-renal como a causa principal de IRA nestes pacientes.

Associação de hipovitaminose D com Lúpus Eritematoso Sistêmico e inflamação

Introdução: Atualmente, é descrita elevada prevalência de hipovitaminose D no Lúpus Eritematoso Sistêmico (LES), a qual se associa a algumas manifestações clínicas e maior atividade inflamatória. Objetivo: Avaliar a associação entre insuficiência de vitamina D com LES e marcadores inflamatórios. Métodos: Estudo transversal, tendo sido avaliados 45 pacientes com LES e 24 controles sem a doença. Níveis de 25-hidroxivitamina D [25(OH)D] menores que 30 ng/mL foram considerados insuficientes. A atividade da doença foi avaliada pelo Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Foram avaliados, ainda, proteína C reativa ultrassensível (PCRus) e interleucina-6 (IL-6) para verificação do status inflamatório. Para avaliação do envolvimento renal, foram realizados análise de elementos anormais e sedimentoscopia urinárias (EAS), hematúria e piúria quantitativas, proteinúria e depuração de creatinina em urina de 24 horas e anti-DNA de dupla hélice sérico. Resultados: A prevalência de insuficiência de 25(OH)D foi de 55% nos pacientes lúpicos e 8% nos participantes controles (p = 0,001). A mediana da 25(OH)D foi menor nos pacientes do que no grupo controle. Os pacientes com insuficiência de 25(OH)D apresentaram níveis mais elevados de IL-6 e maior prevalência de hematúria ao EAS. Não houve correlação entre vitamina D, nefrite lúpica e SLEDAI. Conclusão: Em nosso estudo, a insuficiência de vitamina D foi mais prevalente em pacientes com LES e se associou com níveis mais elevados de IL-6 e presença de hematúria.