Use of lordotic cages in L5-S1 Anterior Lumbar Interbody Fusion (ALIF) procedures

Anterior Lumbar Interbody Fusion (ALIF) has gained popularity in the last few years, thanks to its numerous advantages. Recently the use of lordotic cages has been described, allowing theoretically a better lordosis restoration of the lumbar disc space. We described the results obtained with the use of lordotic cages in 27 patients who underwent ALIF procedure for L5-S1 disc degenerative disease, in terms of segmental lordosis and global lumbar lordosis changes.

Distinguishing the genotype 1 genes and proteins of human Wa-like rotaviruses vs. porcine rotaviruses.

Group A rotaviruses (RVAs) are 11-segmented, double-stranded RNA viruses and important causes of gastroenteritis in the young of many animal species. Previous studies have suggested that human Wa-like RVAs share a close evolutionary relationship with porcine RVAs. Specifically, the VP1-VP3 and NSP2-5/6 genes of these viruses are usually classified as genotype 1 with >81% nucleotide sequence identity. Yet, it remains unknown whether the genotype 1 genes and proteins of human Wa-like strains are distinguishable from those of porcine strains. To investigate this, we performed comprehensive bioinformatic analyses using all known genotype 1 gene sequences. The RVAs analyzed represent wildtype strains isolated from humans or pigs at various geographical locations during the years of 2004-2013, including 11 newly-sequenced porcine RVAs from Brazil. We also analyzed archival strains that were isolated during the years of 1977-1992 as well as atypical strains involved in inter-species transmission between humans and pigs. We found that, in general, the genotype 1 genes of typical modern human Wa-like RVAs clustered together in phylogenetic trees and were separate from those of typical modern porcine RVAs. The only exception was for the NSP5/6 gene, which showed no host-specific phylogenetic clustering. Using amino acid sequence alignments, we identified 34 positions that differentiated the VP1-VP3, NSP2, and NSP3 genotype 1 proteins of typical modern human Wa-like RVAs versus typical modern porcine RVAs and documented how these positions vary in the archival/unusual isolates. No host-specific amino acid positions were identified for NSP4, NSP5, or NSP6. Altogether, the results of this study support the notion that human Wa-like RVAs and porcine RVAs are evolutionarily related, but indicate that some of their genotype 1 genes and proteins have diverged over time possibly as a reflection of sequestered replication and protein co-adaptation in their respective hosts.

Lumbar spinal degenerative "microinstability": hype or hope? Proposal of a new classification to detect it and to assess surgical treatment.

PURPOSE: The stage of unstable dysfunction, also defined as "active discopathy" by Nguyen in 2015 and configuring the first phase of the degenerative cascade described by Kirkaldy-Willis, has specific pathoanatomical and clinical characteristics (low back pain) in the interested vertebral segment, without the presence of spondylolisthesis in flexion-extension radiography. This clinical condition has been defined as "microinstability" (MI). The term has currently not been recognized by the scientific community and is subject of debate for its diagnostic challenge. MI indicates a clinical condition in which the patient has a degeneration of the lumbar spine, causing low back pain, and radiological examinations do not show a spondylolisthesis. METHODS: We elaborated a clinical score test based on preoperative radiological examinations (static and dynamic X-Rays, CT and MRI) to detect and assess MI. Then, we enrolled 74 patients, all the levels from L1 to S1 were analysed, for a total amount of 370 retrospectively analysed levels. We excluded patients with degenerative scoliosis, as it is related to an advanced stage of degeneration. The test has been developed with the aim of furnishing quantitative data on the basis of the aforementioned radiological examinations and of elaborating a diagnosis and a treatment for the degenerative pathology in dysfunctional phase, responsible for low back pain. RESULTS: We performed a statistical analysis on the results obtained from the test in terms of significativity and predictive value with a 1-year follow-up, calculating the p value and the χ (2) value. CONCLUSIONS: In patients with low back pain and negative dynamic X-Rays, an accurate analysis of the radiological exams (CT, MRI, X-Rays) allows to formulate a diagnosis of suspect MI with a good predictive value. This situation opens many clinical and medicolegal scenarios. The preliminary results seem to validate the test with a good predictive value, especially towards ASD, but they need further studies. On the basis of the results obtained, the test seems to allow a good classification of the dysfunctional phase of the degenerative cascade, identifying and classifying MI as a pathologic entity, defining its pathoanatomical and clinical relevance and elaborating a treatment algorithm.

First Case of Systemic Coronavirus Infection in a Domestic Ferret (Mustela putorius furo) in Peru.

A domestic ferret from Lima, Peru, died after ten days of non-specific clinical signs. Based on pathology, immunohistochemistry and molecular analysis, ferret systemic coronavirus (FRSCV)-associated disease was diagnosed for the first time in South America. This report highlights the potential spread of pathogens by the international pet trade.

Betacoronavirus 1 in alpacas (Vicugna pacos) in the High Peruvian Andes.

Genetic sequences highly related to Bovine coronavirus (BCoV) were detected in fecal samples from Peruvian 1-3 week old alpaca crias located on six farms in Puno department, some of which shared pastures with cattle. A total of 60 samples were screened for coronavirus using a nested PCR amplification of a fragment of the RNA-dependent RNA polymerase (RdRp) gene. Sequences from 11 positive samples were highly similar to the Kakegawa, Quebec and Mebus BCoV strains (99.5-100.0%) and 99.2% identical to an alpaca Coronavirus (CoV) previously detected in the USA. The detection of genetic sequences related to BCoV from Peruvian alpaca crias suggests possible role of this virus on enteric disorders etiology in the High Andes.

Description of rotavirus F in broilers from Brazilian poultry farms.

Rotaviruses are segmented double-stranded RNA viruses that cause gastroenteritis in mammals and birds. Here we describe the first partial nucleotide sequences of the structural protein VP6 from the genomes of group F rotaviruses that were detected in 5 out of 53 fecal samples (9.43%) from healthy broilers from Brazilian poultry farms based on reverse-transcriptase-PCR with primers designed for this study. The findings support the development of molecular detection systems, which can be used for the assessment of the distribution of rotavirus F in birds, their potential involvement in diseases, and their impact on poultry health.

Occurrence and characterization of rotavirus A in broilers, layers, and broiler breeders from Brazilian poultry farms.

Rotaviruses are a major cause of diarrhea in humans and animals, including several mammalian and avian species. Using different PCR protocols, we report the occurrence of rotavirus A in 21 (53.84%; 21/39) from 39 fecal pool samples of broilers, layers, and broiler breeders from Brazilian avian farms. We typed the G5, G8, G11, G19, and P[31] genotypes.

First description of group A rotavirus from fecal samples of ostriches (Struthio camelus).

This study investigated the occurrence of rotavirus infections in ostriches (Struthio camelus) reared in Northern Paraná, Brazil. Fecal (n=66) and serum (n=182) samples from nine farms located in four different cities were analyzed by silver stained-polyacrylamide gel electrophoresis (ss-PAGE), RT-PCR assay, virus isolation, and counterimmunoelectroosmophoresis (CIE). Rotavirus group A seropositivity occurred in 5.49% (10/182) of serum samples of ostriches originated from two farms. Only 9.09% (6/66) of fecal samples from ostriches with diarrhea maintained in one farm were positive by ss-PAGE, RT-PCR, and virus isolation. The G (VP7) and P (VP4) genotypes of rotavirus wild strains isolated in cell culture were determined by multiplex-nested PCR. The genotyping identified two rotavirus strains: G6P[1] and G10P[1]. In three rotavirus strains it was only possible to identify the P type; one strain being P[1] and two strains that presented the combination of P[1]+P[7]. These findings might represent the first characterization of rotavirus in ostriches, and the finding of porcine and bovine-like rotavirus genotypes in ostriches might suggest virus reassortment and possible interspecies transmission.

Bovine rotavirus in turkeys with enteritis.

Rotaviruses are the main agents responsible for diarrhea in different animal species and for infantile gastroenteritis. These viruses have been isolated from various avian species and have often been associated with poult enteritis and mortality syndrome. Nevertheless, the knowledge of rotavirus infection in turkeys is scarce. Six group A rotavirus strains obtained from pooled enteric contents of diarrheic turkeys were isolated in MA-104 cell culture and typed as G(6)P(1), a typical bovine rotavirus genotype. Additionally, the electropherotypes showed a migration pattern identical to the Nebraska calf diarrhea virus, and the complete NSP4 gene phylogeny showed that all six strains segregated in the genotype E2. Taken together, these results point toward a cattle-to-turkey rotavirus transmission. As a conclusion, bovine-origin rotavirus can be found in turkeys, and this transmission route must now be considered for the improvement of the health status in turkey farms.

Detecção direta de Actinobacillus pleuropneumoniae em órgãos de suídeos do estado de São Paulo pela técnica de reação em cadeia pela polimerase (nested-PCR) / Direct detection of Actinobacillus pleuropneumoniae in suidae organs in São Paulo state, Brazil, by polymerase chain reaction (nested-PCR)

A pleuropneumonia suína, causada pelo Actinobacillus pleuropneumoniae, é uma importante doença respiratória, responsável por prejuízos e queda de produtividade nas criações. Este trabalho teve como objetivo determinar a ocorrência de A. pleuropneumoniae em amostras de campo, mediante a adaptação e emprego de uma técnica de nested-PCR dirigida ao gene Apx IV. Definiu-se a sensibilidade analítica das técnicas de PCR e nested-PCR utilizando a amostra padrão A. pleuropneumoniae sorotipo III, em concentrações de DNA variando entre 30 µg/mL a 0,01 ng/ mL. Um total de trinta e sete amostras de campo encaminhadas ao Instituto Biológico entre 1995 a 2007 foram analisadas pelas técnicas de PCR e nested-PCR. A avaliação da sensibilidade analítica revelou que a PCR possui capacidade de gerar sinal a partir de 2 ng/mL de DNA extraído e a nested-PCR a partir de 0,4 ng/mL. Uma vez que a nested-PCR apresentou sensibilidade analítica cinco vezes maior se comparada à PCR para detecção de A. pleuropneumoniae em amostra padrão, o seu emprego pode minimizar a ocorrência de resultados tipo "falso-negativo". Dentre as amostras testadas, dez foram positivas à nested-PCR, sendo observada a ocorrência de A. pleuropneumoniae em nove diferentes animais, um deles javali. A presente técnica de nested-PCR pode ser utilizada para detecção direta de A. pleuropneumoniae em amostras de campo, mesmo após congelamento da amostra por longos períodos e sem necessidade de isolamento bacteriano prévio.

Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae, is an important respiratory disease, responsible for economic losses and reduced productivity. The aim of this study was to determine occurrence of A. pleuropneumoniae in field samples, using an adapted nested-PCR reaction targeting the Apx IV gene. Different DNA concentrations (from 30 µg/mL to 0.01 ng/mL) of A. pleuropneumoniae serotype III reference strain were used to determine the level of sensitivity of first generation and nested-PCR reactions. Thirty-seven field samples sent to Instituto Biológico from 1995 to 2007 were tested by PCR and nested-PCR. Determination of the level of sensitivity showed that PCR could amplify to 2 ng/mL of extracted DNA and nested-PCR to 0.4 ng/mL. Since the nested reaction exhibited a level of sensitivity 5 times greater than the PCR reaction to detect a reference strain, using nested-PCR could minimize the occurrence of false-negative results. Among tested samples, 10 of them were nested-PCR positive, showing occurrence of A. pleuropneumoniae in 9 different animals (including one wild boar). This nested-PCR reaction can be used for direct detection of A. pleuropneumoniae in field samples, even after frozen storage for long periods, without the need for previous bacterial isolation.

Identification of Porcine parvovirus from wild boars by partial sequencing of the VP-2 coding gene / Identificação de parvovírus suíno em javalis a partir do sequenciamento parcial do gene VP-2

Este estudo descreve a detecção e a identificação de DNA de parvovírus suíno (PVS) em amostras de órgãos de dois javalis, por PCR e sequenciamento direcionado ao gene VP-2. Pools de órgãos (baço, rins, fígado, linfonodos e tonsila) de três javalis adultos e assintomáticos de Paraguaçu Paulista, SP, criados com propósitos comerciais, foram submetidos à detecção de PVS, resultando em duas amostras positivas após reações de nested-PCR direcionadas aos genes NS-1 e VP-2. Os fragmentos parciais de VP-2 foram sequenciados e comparados a sequências homólogas de cepas NADL-2 e Kresse, demonstrando identidade nucleotídica de 100%. Com relação a 29 cepas de PVS previamente isoladas no Brasil, o grau de identidade nucleotídica variou de 99 a 100% (uma a três substituições de nucleotídeos). Estes resultados demonstram, pela primeira vez, a detecção direta por PCR de parvovírus suíno em javalis, confirmada por análise de sequenciamento genético.

On the etiology of an outbreak of winter dysentery in dairy cows in Brazil

Winter dysentery (WD) is a seasonal infectious disease described worldwide that causes a marked decrease in milk production in dairy cows. In the Northern hemisphere, where the disease is classically recognized, bovine coronavirus (BCoV) has been assigned as a major etiologic agent of the disease. Nonetheless, in the Southern hemisphere, an in-deep etiological survey on WD cases had not been carried out. This study aimed to survey for BCoV by nested-RT-PCR, rotavirus by polyacrylamide gel electrophoresis (PAGE) and ELISA, bacteria by classical bacteriological methods and PCR for virulence factors and parasites by sugar flotation test on fecal samples of 21 cows from a farm during an outbreak of WD in São Paulo state, Southeastern Brazil. BCoV was detected in all 21 samples, while rotavirus was detected in two symptomatic cows. Escherichia coli, Yersinia intermedia, Providencia rustigianii Proteus penneri, Klebsiella terrigena and Enterobacter aglomerans were detected in samples from both asymptomatic and healthy cows in different associations. The study of E. coli virulence factors revealed that the strains isolated were all apathogenic. Cysts of Eimeria sp. and eggs of Strongyloidea were detected at low numbers in four of the symptomatic cows, with one co-infestation. These results suggest BCoV as the main etiologic agent of the cases of WD in Brazil, a conclusion that, with the clinical and epidemiological patterns of the disease studied herein, match those already described elsewhere. These findings give basis to the development of preventive measures and contribute to the understanding of the etiology of WD.

Em vacas leiteiras, a disenteria de inverno (DI) é uma doença infecciosa sazonal mundialmente relatada que ocasiona uma marcada queda na produção de leite; no hemisfério Norte, onde a doença é classicamente reconhecida, o coronavirus bovino (BCoV) tem um importante papel como agente etiológico. Entretanto, no hemisfério Sul, pesquisas etiológicas aprofundadas em casos de DI nunca forma realizadas. Este estudo objetivou a pesquisa de BCoV utilizando nested-RT-PCR, rotavírus utilizando eletroforese em gel de poliacrilamida (PAGE) e ELISA, bactérias com métodos bacteriológicos clássicos e PCR para fatores de virulência e parasitas pela técnica de flutuação em açúcar em 21 amostras fecais de vacas de uma fazenda durante um surto de DI no estado de São Paulo, Sudeste do Brasil. BCoV foi encontrado em todas as 21 amostras, enquanto que rotavírus foi encontrado em duas vacas sintomáticas. Escherichia coli, Yersinia intermedia, Providencia rustigiani, Proteus penneri, Klebsiella terrigena e Enterobacter aglomerans foram encontradas tanto em amostras de vacas sintomáticas quanto assintomáticas. O estudo de fatores de virulência para E. coli revelou que as amostras isoladas eram todas apatogênicas. Cistos de Eimeria sp. e ovos de Strongyloidea foram encontrados em baixos números em quatro animais sintomáticos, com uma co-infestação. Tais resultados sugerem o BCoV como o principal agente etiológico em casos de DI no Brasil, uma conclusão que, somada aos padrões clínicos e epidemiológicos da doença aqui estudada, concordam com aqueles descritos em outras regiões. Estes achados fornecem base o desenvolvimento de medidas preventivas e também contribuem para o entendimento sobre a etiologia da DI.

Winter dysentery in cows associated with Bovine Coronavirus (BCoV)

Descreve-se a pesquisa de BCoV e rotavírus em 13 mostras fecais de vacas de surtos de disenteria utilizando uma nested PCR dirigida ao gene RdRp e PAGE, respectivamente. Todas as amostras fecais foram positivas para BCoV e nenhuma delas apresentou-se positiva para rotavírus em PAGE. O encontro de coronavírus bovino em amostras fecais de vacas com disenteria sugere que este vírus possa ser o agente primário envolvido na etiologia dos casos aqui relatados

Diagnosis of canine brucellosis: comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer.

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Phylogenetic analysis of Anaplasma marginale strains from Paraná State, Brazil, using the msp1alpha and msp4 genes.

Anaplasma marginale is an obligate intraerythrocytic rickettsial pathogen (order, Rickettsiales: family, Anaplasmataceae) that causes bovine anaplasmosis. This disease is widely distributed in tropical and sub-tropical regions of the world and causes important economic losses to cattle production. Major surface protein (MSP)1a (msp1alpha gene) is one of the six MSPs identified on A. marginale from cattle, whose sequence and size vary according to the number of tandem 28- to 29-amino acid repeats. This study characterized the msp1alpha and msp4 genes obtained from three distinct Brazilian herds from the State of Paraná. Three strains of the msp1alpha and one strain of the msp4 gene were sequenced. The strains evaluated revealed PCR products of different size, representing three, five and six internal repeats. Sequence analyses confirmed the number of tandem sequence copies and revealed a high degree of sequence identity with strains from other Brazilian States, as well as strains from the USA, Europe and Israel. The msp1alpha DNA and amino acid sequences from A. marginale and DNA sequences of msp4 strains did not reveal distinct phylogeographical segregation. However, the amino acid sequences of msp4 demonstrated definite phylogeographical relationship. These results suggest that the amino acid sequences of msp4 should be used for phylogenetic identification of A. marginale strains and may be an important tool for the epidemiology and control of anaplasmosis. Additionally, the close similarity of the Paraná strains of A. marginale with strains from USA, Europe and Asia may reflect the introduction of these genes during the development of the Brazilian bovine herd.

Molecular analysis of Brazilian strains of bovine coronavirus (BCoV) reveals a deletion within the hypervariable region of the S1 subunit of the spike glycoprotein also found in human coronavirus OC43.

Bovine coronavirus (BCoV) causes enteric and respiratory dis- orders in calves and dysentery in cows. In this study, 51 stool samples of calves from 10 Brazilian dairy farms were analysed by an RT-PCR that amplifies a 488-bp fragment of the hypervariable region of the spike glycoprotein gene. Maximum parsimony genealogy with a heuristic algorithm using sequences from 15 field strains studied here and 10 sequences from GenBank and bredavirus as an outgroup virus showed the existence of two major clusters (1 and 2) in this viral species, the Brazilian strains segregating in both of them. The mean nucleotide identity between the 15 Brazilian strains was 98.34%, with a mean amino acid similarity of 98%. Strains from cluster 2 showed a deletion of 6 amino acids inside domain II of the spike protein that was also found in human coronavirus strain OC43, supporting the recent proposal of a zoonotic spill- over of BCoV. These results contribute to the molecular characterization of BCoV, to the prediction of the efficiency of immunogens, and to the definition of molecular markers useful for epidemiologic surveys on coronavirus-caused diseases.

A nested polymerase chain reaction test for bovine coronavirus diagnosis bases on the RNA-dependent RNA-polymerase gene

Bovine coronavirus (BCoV) causes enteritis and respiratory iIIness in calves and dysentery in cows. Difficulties found in the production of large amounts of BCoV in cel! culture and those regarding envelope integrity pose a major problem to antigen, hyperimmune sera and monoclonal antibodies production. This study aimed at the development of a polymerase chain reaction for BCoV detection in fecal samples of calves targeted to the gene of the RNA-dependent RNApolymerase, and the comparison of this test with the hemagglutination/ hemagglutination inhibition test (HA/HI). Both tests were applied to 203 fecal samples. HA/HI resulted in an individual-levei BCoV frequency of 35.47% and a farm-Ievel frequency of 73.68%, while the proposed PCR resulted in an individual-levei BCoV frequency of 25.12% and a farm-Ievel frequency of 52.63%. Both Kappa and Youden's tests revealed a low agreement between PCR and HA/HI. Hypotheses are suggested to explain such a disagreement. The PCR proposed is a useful toei forthe detection of BCoV, for epidemiological surveys and as a screening test for studies focused on other BCoV genes, such as the spike gene.

Estudio comparativo de quinapril frente a la combinación de quinapril e hidroclorotiazida en el tratamiento de la hipertensión arterial ligera-moderada. Réplica / Comparative study of quinapril versus quinapril/hydrochlorothiazide in the treatment of mild-to-moderate hypertension. Reply

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