Rehabilitation interventions in long COVID: a case report / Intervenções em reabilitação na COVID longa: relato de caso

ABSTRACT. The repercussions of Long COVID demand specialised rehabilitation care. Interdisciplinary interventions in a 58-year-old patient were implemented to improve the clinical, motor and cognitive complaints associated with COVID-19. The rehabilitation team performed quantitative and qualitative evaluations in the initial phase and after 12 months of follow-up. The patient's neuropathic pain, ankle and foot muscle strength, gait pattern, general cognitive functioning, initiative, emotional expressiveness, processing speed, neuropsychiatric symptoms and quality of life improved. She demonstrated gains in metacognition and expanded the use of compensatory strategies, resuming her routine and professional activities, although still with signs of executive dysfunction. It is concluded that a rehabilitation program calibrated to the profile of the patient with Long COVID had positive effects on functionality and satisfaction with quality of life.

RESUMO. As repercussões da COVID Longa demandam atendimentos especializados de reabilitação. Intervenções interdisciplinares em paciente de 58 anos foram implementadas com o objetivo de melhorar as queixas clínicas, motoras e cognitivas associadas à COVID-19. A equipe de reabilitação realizou avaliações quantiqualitativas na fase inicial e após 12 meses de acompanhamento. A paciente evoluiu com melhora da dor neuropática, da força muscular de tornozelo e pé, do padrão de marcha, do funcionamento cognitivo geral, da iniciativa, da expressividade emocional, da velocidade de processamento, dos sintomas neuropsiquiátricos e da qualidade de vida (domínios físico e psicológico). Demonstrou ganhos na metacognição e ampliou o uso de estratégias compensatórias, retomando suas atividades rotineiras e profissionais, embora ainda com indícios de disfunção executiva. Conclui-se que um programa de reabilitação calibrado ao perfil do paciente com COVID longa trouxe efeitos positivos na funcionalidade e satisfação com a qualidade de vida.

Performance of PRISM III and PIM 2 scores in a cancer pediatric intensive care unit. / Desempenho do PRISM III e do PIM 2 em unidade de terapia intensiva pediátrica oncológica.

OBJECTIVE: To assess the performance of Pediatric Risk of Mortality (PRISM) III and Pediatric Index of Mortality (PIM) 2 scores in the pediatric intensive care unit. METHODS: A retrospective cohort study. Data were retrospectively collected from medical records of all patients admitted to the pediatric intensive care unit of a cancer hospital from January 2017 to June 2018. RESULTS: The mean PRISM III score was 15, and PIM 2, 24%. From the 338 studied patients, 62 (18.34%) died. The PRISM III estimated mortality was 79.52 patients (23.52%) and for PIM 2 80.19 patients (23.72%), corresponding to a standardized mortality ratio (95% confidence interval: 0.78 for PRISM II and 0.77 for PIM 2). The Hosmer-Lemeshow chi-square test was 11.56, 8df, 0.975 for PRISM II and 0.48, 8df, p = 0.999 for PIM 2. The area under the Receiver Operating Characteristic curve was 0.71 for PRISM III and 0.76 for PIM 2. CONCLUSION: Both scores overestimated mortality and have shown a regular ability to discriminate between survivors and non-survivors. Models should be developed to quantify the severity of cancer pediatric patients in Pediatric Intensive Care Units and to predict the mortality risk accounting for their peculiarities.

OBJETIVO: Avaliar o desempenho do Pediatric Risk of Mortality (PRISM) III e do Pediatric Index of Mortality (PIM) 2 em unidade de terapia intensiva pediátrica. MÉTODOS: Estudo de coorte retrospectivo. Os dados retrospectivos foram coletados dos prontuários de todos os pacientes admitidos na unidade de terapia intensiva pediátrica de um hospital infantil oncológico, entre janeiro de 2017 a junho de 2018. RESULTADOS: A média do PRISM III foi de 15 e do PIM 2 de 24%. Dos 338 pacientes estudados, 62 (18,34%) morreram. A mortalidade estimada pelo PRISM III foi de 79,52 (23,52%) e pelo PIM 2 de 80,19 (23,72%) pacientes, correspondendo a taxa padronizada de mortalidade (intervalo de confiança de 95%) de 0,78 para o PRISM II e 0,77 para o PIM 2. O teste de ajuste de Hosmer-Lemeshow obteve qui-quadrado de 11,56, 8df, com p = 0,975, para PRISM III, e qui-quadrado de 0,48, 8df, p = 0,999, para o PIM 2. Foi obtida área sob a curva Característica de Operação do Receptor de 0,71 para o PRISM III e 0,76 para o PIM 2. CONCLUSÃO: Os dois escores superestimaram a mortalidade e demonstraram poder regular de discriminação entre sobreviventes e não sobreviventes. Devem ser desenvolvidos modelos para quantificar a gravidade de pacientes pediátricos com câncer em unidade de terapia intensiva pediátrica e predizer o risco de mortalidade que contemplem suas peculiaridades.

Desempenho do PRISM III e do PIM 2 em unidade de terapia intensiva pediátrica oncológica / Performance of PRISM III and PIM 2 scores in a cancer pediatric intensive care unit

RESUMO Objetivo: Avaliar o desempenho do Pediatric Risk of Mortality (PRISM) III e do Pediatric Index of Mortality (PIM) 2 em unidade de terapia intensiva pediátrica. Métodos: Estudo de coorte retrospectivo. Os dados retrospectivos foram coletados dos prontuários de todos os pacientes admitidos na unidade de terapia intensiva pediátrica de um hospital infantil oncológico, entre janeiro de 2017 a junho de 2018. Resultados: A média do PRISM III foi de 15 e do PIM 2 de 24%. Dos 338 pacientes estudados, 62 (18,34%) morreram. A mortalidade estimada pelo PRISM III foi de 79,52 (23,52%) e pelo PIM 2 de 80,19 (23,72%) pacientes, correspondendo a taxa padronizada de mortalidade (intervalo de confiança de 95%) de 0,78 para o PRISM II e 0,77 para o PIM 2. O teste de ajuste de Hosmer-Lemeshow obteve qui-quadrado de 11,56, 8df, com p = 0,975, para PRISM III, e qui-quadrado de 0,48, 8df, p = 0,999, para o PIM 2. Foi obtida área sob a curva Característica de Operação do Receptor de 0,71 para o PRISM III e 0,76 para o PIM 2. Conclusão: Os dois escores superestimaram a mortalidade e demonstraram poder regular de discriminação entre sobreviventes e não sobreviventes. Devem ser desenvolvidos modelos para quantificar a gravidade de pacientes pediátricos com câncer em unidade de terapia intensiva pediátrica e predizer o risco de mortalidade que contemplem suas peculiaridades.

ABSTRACT Objective: To assess the performance of Pediatric Risk of Mortality (PRISM) III and Pediatric Index of Mortality (PIM) 2 scores in the pediatric intensive care unit. Methods: A retrospective cohort study. Data were retrospectively collected from medical records of all patients admitted to the pediatric intensive care unit of a cancer hospital from January 2017 to June 2018. Results: The mean PRISM III score was 15, and PIM 2, 24%. From the 338 studied patients, 62 (18.34%) died. The PRISM III estimated mortality was 79.52 patients (23.52%) and for PIM 2 80.19 patients (23.72%), corresponding to a standardized mortality ratio (95% confidence interval: 0.78 for PRISM II and 0.77 for PIM 2). The Hosmer-Lemeshow chi-square test was 11.56, 8df, 0.975 for PRISM II and 0.48, 8df, p = 0.999 for PIM 2. The area under the Receiver Operating Characteristic curve was 0.71 for PRISM III and 0.76 for PIM 2. Conclusion: Both scores overestimated mortality and have shown a regular ability to discriminate between survivors and non-survivors. Models should be developed to quantify the severity of cancer pediatric patients in Pediatric Intensive Care Units and to predict the mortality risk accounting for their peculiarities.

Microcephaly in north-east Brazil: a retrospective study on neonates born between 2012 and 2015.

OBJECTIVE: To assess the number of children born with microcephaly in the State of Paraíba, north-east Brazil. METHODS: We contacted 21 maternity centres belonging to a paediatric cardiology network, with access to information regarding more than 100 000 neonates born between 1 January 2012 and 31 December 2015. For 10% of these neonates, nurses were requested to retrieve head circumference measurements data from delivery-room books. We used three separate criteria to classify whether a neonate had microcephaly: (i) the Brazilian Ministry of Health proposed criterion: term neonates (gestational age ≥ 37 weeks) with a head circumference of less than 32 cm; (ii) Fenton curves: neonates with a head circumference of less than -3 standard deviation for age and gender; or (iii) the proportionality criterion: neonates with a head circumference of less than ((height/2))+10) ± 2. FINDINGS: Between 1 and 31 December 2015, nurses obtained data for 16 208 neonates. Depending on which criterion we used, the number of neonates with microcephaly varied from 678 to 1272 (4.2-8.2%). Two per cent (316) of the neonates fulfilled all three criteria. We observed temporal fluctuations of microcephaly prevalence from late 2012. CONCLUSION: The numbers of microcephaly reported here are much higher than the 6.4 per 10 000 live births reported by the Brazilian live birth information system. The results raise questions about the notification system, the appropriateness of the diagnostic criteria and future implications for the affected children and their families. More studies are needed to understand the epidemiology and the implications for the Brazilian health system.

Variation in lipid extractability by solvent in microalgae. Additional criterion for selecting species and strains for biofuel production from microalgae.

The lipid extractability of 14 microalgae species and strains was assessed using organic solvents (methanol and chloroform). The high variability detected indicated the potential for applying this parameter as an additional criterion for microalgae screening in industrial processes such as biofuel production from microalgae. Species without cell walls presented higher extractability than species with cell walls. Analysis of cell integrity by flow cytometry and staining with propidium iodide showed a significant correlation between higher resistance to the physical treatments of cell rupture by sonication and the lipid extractability of the microalgae. The results highlight the cell wall as a determining factor in the inter- and intraspecific variability in lipid extraction treatments.

Mechanisms involved in quinolone resistance in Mycoplasma mycoides subsp. capri.

Mycoplasma mycoides subsp. capri is a causative agent of contagious agalactia in goats. In this study, M. mycoides subsp. capri mutants were selected for resistance to fluoroquinolones (norfloxacin, enrofloxacin and ciprofloxacin) by serial passes in broth with increasing concentrations of antibiotic. Mutations conferring cross-resistance to the three fluoroquinolones were found in the quinolone resistance determining regions of the four genes encoding DNA gyrase and topoisomerase IV. Different mutations in the DNA gyrase GyrA subunit suggest a different mechanism of inhibition between norfloxacin and the other tested fluoroquinolones. The presence of an adenosine triphosphate-dependent efflux system was suggested through the use of the inhibitor orthovanadate.

Molecular taxonomy of Dunaliella (Chlorophyceae), with a special focus on D. salina: ITS2 sequences revisited with an extensive geographical sampling.

We used an ITS2 primary and secondary structure and Compensatory Base Changes (CBCs) analyses on new French and Spanish Dunallela salina strains to investigate their phylogenetic position and taxonomic status within the genus Dunaliella. Our analyses show a great diversity within D. salina (with only some clades not statistically supported) and reveal considerable genetic diversity and structure within Dunaliella, although the CBC analysis did not bolster the existence of different biological groups within this taxon. The ITS2 sequences of the new Spanish and French D. salina strains were very similar except for two of them: ITC5105 "Janubio" from Spain and ITC5119 from France. Although the Spanish one had a unique ITS2 sequence profile and the phylogenetic tree indicates that this strain can represent a new species, this hypothesis was not confirmed by CBCs, and clarification of its taxonomic status requires further investigation with new data. Overall, the use of CBCs to define species boundaries within Dunaliella was not conclusive in some cases, and the ITS2 region does not contain a geographical signal overall.

Effects on goat milk quality of the presence of Mycoplasma spp. in herds without symptoms of contagious agalactia.

This study was designed to assess the possible effects of mycoplasmas on the quality of milk produced by goat herds in a contagious agalactia (CA) endemic area with absence of classical symptoms. Several factors related to milk quality (percentages of fat, total protein, lactose and total solids, standard plate counts (SPC) and presence of Staphylococcus aureus) were compared in mycoplasma-infected and non-infected herds. To define the CA status of 26 herds on the island of Lanzarote (Spain), where CA is endemic, 570 individual milk samples and 266 bulk tank milk (BTM) samples were microbiologically analysed for the presence of Mycoplasma spp. A herd was considered infected by mycoplasmas when at least a sample (individual or BTM) was positive. BTM samples were also used to determine milk quality parameters. Mycoplasma infection was confirmed in 13 herds. A total of 31, 10 and 11 strains of Mycoplasma mycoides subsp. mycoides LC (MmmLC), Mp. agalactiae and Mp. capricolum subsp. capricolum were isolated. No significant differences were observed between the least square means of the variables fat, total protein, lactose and total solids or SPC recorded for the infected v. non-infected herds. The Staph. aureus status of a herd was also found to be independent of the presence of Mycoplasma spp. Our findings indicate that neither the presence of mycoplasmas in a goat herd with absence of classical symptoms seem to compromise the quality of the BTM.

Utilização de medicamentos durante a gravidez na cidade de Natal, Rio Grande do Norte, Brasil / Drug use during pregnancy in Natal, Brazil

OBJETIVO: estudar o uso de medicamentos por gestantes atendidas durante o pré-natal em unidades básicas do Sistema Único de Saúde (SUS) na cidade de Natal, rio Grande do Norte, Brasil. MÉTODOS: foram entrevistadas 610 grávidas, entre o primeiro e o terceiro trimestre de gestação, que compareceram para consulta pré-natal em unidades de saúde localizadas nos quatro distritos sanitários de Natal, entre maio e julho de 2006. Os dados foram coletados com entrevistas estruturadas, baseando-se em perguntas uso-orientadas e medicamento-orientadas. Os fármacos foram classificados de acordo com o Anatomical Therapeutic Chemical Classification System (ATC) e segundo critérios de risco para a gestação da Food and Drug Administration (FDA). Utilizou-se teste do chi2 para análise dos dados. RESULTADOS: eram utilizados 1.505 medicamentos, obtendo-se uma média de 2,4 drogas por mulher. O uso de pelo menos um fármaco na gravidez foi relatado por 86,6 por cento das gestantes. As classes mais utilizadas foram os antianêmicos (35,6 por cento dos medicamentos), analgésicos (24,9 por cento), drogas para distúrbios gastrintestinais (9,1 por cento) e vitaminas (7 por cento). De acordo com a classificação do FDA, dos medicamentos empregados 42,7 por cento pertencem a categoria A de risco; 27,1 por cento à categoria B, 29,3 por cento à categoria C; 0,3 à categoria D e nenhum à categoria X. Foram usados, no primeiro trimestre da gestação, 43,6 por cento dos fármacos. Observou-se maior uso de medicamentos quanto maior a escolaridade e a renda familiar da mulher. A automedicação ocorreu em 12,2 por cento dos medicamentos; esse índice foi maior no primeiro trimestre de gravidez e em gestantes de baixa escolaridade e multigestas. CONCLUSÕES: as gestantes de Natal estão sendo expostas a uma variedade de medicamentos, cuja segurança na gravidez ainda é incerta, o que exige prescrição criteriosa para evitar possíveis danos ao feto.

PURPOSE: to study the use of medicines by pregnant women during prenatal care in clinics of the national public health system in the city of Natal, Brazil. METHODS: a total of 610 pregnant women between the first and the third trimesters of pregnancy were interviewed in the public clinics of the four sanitary districts of Natal, from May to July 2006. The data were collected by a structured questionnaire, based in use-oriented and medicine-oriented questions. The drugs were classified according to the Anatomical Therapeutic Chemical Classification System (ATC), in agreement with the gestation risk criteria from the Food and Drugs Administration (FDA). The statistical analysis was made by the chi2 test. RESULTS: a total of 1,505 drugs were used, with an average of 2.4 medications per woman. The use of at least one drug was found in 86.6 percent of the women. The most frequently used drugs were anti-anemics (35.6 percent), analgesics (24.9 percent), drugs for gastrointestinal disorders (9.1 percent) and vitamins (7 percent). According to the FDA classification, 42.7 percent belonged to category A risk, 27.1 percent to category B, 29.3 percent to category C, 0.3 percent to category D and none to category X. The use of medicines during the first trimester of pregnancy amounted to 43.6 percent. The rate of drug use increased with higher schooling level and family income. Self-medication was found in 12.2 percent of the drug intake and this rate was higher in the first trimester of gestation and with women with low education level and previous gestations. CONCLUSIONS: pregnant women from Natal are being exposed to a variety of medicines with uncertain safety in pregnancy. Therefore, more careful prescription is needed, to avoid possible fetal damage.

[Drug use during pregnancy in Natal, Brazil]. / Utilização de medicamentos durante a gravidez na cidade de Natal, Rio Grande do Norte, Brasil.

PURPOSE: to study the use of medicines by pregnant women during prenatal care in clinics of the national public health system in the city of Natal, Brazil. METHODS: a total of 610 pregnant women between the first and the third trimesters of pregnancy were interviewed in the public clinics of the four sanitary districts of Natal, from May to July 2006. The data were collected by a structured questionnaire, based in use-oriented and medicine-oriented questions. The drugs were classified according to the Anatomical Therapeutic Chemical Classification System (ATC), in agreement with the gestation risk criteria from the Food and Drugs Administration (FDA). The statistical analysis was made by the chi2 test. RESULTS: a total of 1,505 drugs were used, with an average of 2.4 medications per woman. The use of at least one drug was found in 86.6% of the women. The most frequently used drugs were anti-anemics (35.6%), analgesics (24.9%), drugs for gastrointestinal disorders (9.1%) and vitamins (7%). According to the FDA classification, 42.7% belonged to category A risk, 27.1% to category B, 29.3% to category C, 0.3% to category D and none to category X. The use of medicines during the first trimester of pregnancy amounted to 43.6%. The rate of drug use increased with higher schooling level and family income. Self-medication was found in 12.2% of the drug intake and this rate was higher in the first trimester of gestation and with women with low education level and previous gestations. CONCLUSIONS: pregnant women from Natal are being exposed to a variety of medicines with uncertain safety in pregnancy. Therefore, more careful prescription is needed, to avoid possible fetal damage.

Detection of mycoplasmas in goat milk by flow cytometry.

The detection of mycoplasma in milk can be performed by either culture techniques or polymerase chain reaction (PCR) based methods. Although PCR can reduce the average diagnostic time to 5 h in comparison with the several days for the isolation of the agent, there is still a need to develop methods, which could give earlier results. For this purpose, we tested the ability of flow cytometry (FC) to detect mycoplasmas in milk samples. Milk samples inoculated with four different mycoplasmas, Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. Capricolum, or Mycoplasma mycoides subsp. mycoides large-colony type, known to cause contagious agalactia in goats, were stained with the DNA stain SYBR Green I and analyzed by FC. Three goat milk samples, from which mycoplasmas have been isolated in broth medium were also analyzed. All mycoplasmas were easily distinguished from debris of milk samples, but it was not possible to distinguish between the different mycoplasma species. In our conditions, the detection limit of the technique was of the order of 10(3)-10(4) cells ml(-1). Furthermore, mycoplasmas were also distinguished from Staphylococcus aureus. FC together with SYBR Green I was able to distinguish between mycoplasma cells and debris present in milk samples and gave results in 20-30 min. This is an important first step in developing a robust, routine flow cytometric method for the detection of mycoplasmas in milk samples.

Field trial of two dual vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) in goats.

Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (LC type) were developed using inactivated strains selected in previous characterization studies. The vaccines differed in terms of the adjuvants used: aluminium hydroxide (vaccine A) or aluminium hydroxide plus purified saponin (vaccine B). These vaccines were tested on 60 pregnant goats and 60 seronegative kids that were challenged by placing in a herd with a history of caprine contagious agalactia (CCA). Our findings indicate the effectiveness of the vaccines in preventing the appearance of new clinical signs such as mastitis, abortion, pneumonia and polyarthritis in CCA affected herds.

Field trial of a combined vaccine against caprine contagious agalactia: humoral immune response in lactating goats.

Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) were developed using inactivated strains selected in previous characterization studies. Formaldehyde and phenol were used as the inactivating agents for vaccines A and B, respectively. Aluminium hydroxide plus purified saponin (Quil-A) were added to both vaccines as adjuvant. The field trial was designed to evaluate the specific humoral immune response to the two mycoplasma species in lactating goats over a period of 7 months. The vaccines were tested on 120 goats randomly assigned to three groups of 40 animals each. Two groups received two injections of vaccine A or B respectively, and a third group remained in the herd as control. Antibody titres determined by ELISA indicated a significant difference between both vaccines and the control group over a 6-month period. Immunoblotting assays also revealed the production of antibodies against the two mycoplasma species. Further field trials are underway to evaluate the efficacy and protection conferred to the animals by these specific antibodies.

Applications of flow cytometry to mycoplasmology.

Flow cytometry has become a valuable tool in different fields of microbiology, such as clinical microbiology, aquatic and environmental microbiology, food microbiology, and biotechnology. It combines direct and rapid assays to determine numbers, biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population. This review focuses on the applications of flow cytometry to the field of mycoplasmology. It tries to give a scope of the important breakthroughs which occurred in this field in the last decades, and in the advantages of introducing flow cytometry in research and routine diagnostic procedures of mycoplasmas.

Flow cytometric method for the assessment of the minimal inhibitory concentrations of antibacterial agents to Mycoplasma agalactiae.

In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MIC) of seven antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, oxytetracycline, and tylosin) on Mycoplasma (M.) agalactiae. Flow cytometry was able to detect M. agalactiae inhibition from 6 h postincubation, although it seems that definitive MIC values determined by flow cytometry were only possible at 12-h postincubation. However, the results obtained by the traditional method were only obtained at 24 h, when a visible change in the medium had occurred. At 24 h, both methods gave the same result for six antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, and oxytetracycline); whereas flow cytometry gave slightly higher MIC for tylosin. This was attributed to the fact that the M. agalactiae growth that had occurred in the tubes containing tylosin was not enough to visibly change the color of the medium. Futhermore, flow cytometry detected that inhibitory concentrations of oxytetracycline, chloramphenicol, and tylosin as judged at 24 h were not able to inhibit the M. agalactiae growth after 48 h. MIC values of enrofloxacin and ciprofloxacin were sufficient only to maintain the total counts per milliliter throughout the time matched samples, whereas higher concentrations of theses antibacterial agents reduced the total counts per milliliter over the course of the experiment. The main advantage of the flow cytometric method is that MIC results for M. agalactiae can be obtained in a shorter time than is possible with the traditional method. The method presented makes identification of resistant populations of M. agalactiae possible and, unlike the traditional method, allows the effect of each antibacterial agent to be determined in real-time at the single-cell level.

Flow cytometric determination of the effects of antibacterial agents on Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. capricolum, and Mycoplasma mycoides subsp. mycoides large colony type.

Flow cytometry together with SYBR green I and propidium iodide was used to study the effects of enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and tylosin on four mycoplasma species. Inhibition of mycoplasma growth could be detected by as early as 3 h after the start of treatment. The strongest effect was observed with enrofloxacin- and ciprofloxacin-treated cells.

Quantification of mycoplasmas in broth medium with sybr green-I and flow cytometry.

Mycoplasmas are the smallest and simplest organisms known. They form a large group of bacteria that can infect humans, animals, and plants. Even though several techniques have been proposed to enumerate mycoplasmas in broth medium, the determination of mycoplasma growth still remains a difficult task. The potential of using flow cytometry (FC) for rapidly estimating several species of mycoplasmas, M. agalactiae (Ma), M. putrefaciens (Mp), M. capricolum subsp. capricolum (Mcc), M. bovis (Mb), M. capricolum subsp. capripneumoniae (Mccp) and M. hyopneumoniae (Mh) in broth medium was examined. The FC analysis was performed by staining the mycoplasma cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count (Colony Forming Units--CFU) or Colour Changing Units (CCU) methods, depending on the mycoplasma species. There was a good correlation between mycoplasma counts determined by FC (cells ml(-1)) and by traditional plate count (CFU) or CCU methods. A correlation of 0.841, 0.981, 0.960, 0.913, 0.954, and 0.844 was obtained for Ma, Mp, Mcc, Mb, Mccp and Mh, respectively. FC method allowed results in 20-30 min, while 24-72 h was necessary for plate count method and 15 days for CCU method. FC was found to be a very useful, practical and fast technique to count mycoplasmas. These findings suggest that FC can be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasmas in broth medium.

Cytokine expression in porcine lungs experimentally infected with Mycoplasma hyopneumoniae.

To gain further insight into the pathogenesis of porcine enzootic pneumonia (PEP), cytokine expression in different pulmonary compartments was examined. Mycoplasma hyopneumoniae (Mh) and proinflammatory and immunoregulatory cytokines (IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-alpha) were detected by immunohistochemical methods in porcine lungs experimentally infected with Mh. Ten pigs were inoculated intranasally with Mh and killed in pairs weekly from 1- to 5-week post-inoculation (wpi). Three Mh-free pigs were taken as controls. Mh-antigen was shown in paraffin-wax-embedded tissues by immunohistochemistry in the luminal surface of bronchial and bronchiolar epithelial cells of all Mh-infected pigs. Significant increase in cytokine expression was detected on snap-frozen tissues from the bronchoalveolar exudate of the airways, mononuclear cells of the alveolar septa and macrophages and lymphocytes of the peribronchial and peribronchiolar lymphoid tissue, from 1 wpi onwards, compared to expression in non-pneumonic lungs. The main cytokines in the BALT of Mh-infected animals that showed an increase were IL-2, IL-4, IL-8, IL-10 and TNF-alpha. In the alveolar septa and bronchoalveolar exudate IL-1 (alpha and beta), IL-2, IL-4, IL-8 and IL-10 expression also increased in infected animals.

Humoral inmune response in lambs and goat kids inoculated with a dual vaccine against contagious agalactia

Una vacuna polivalente frente a Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (largecolony) fue elaborada utilizando cepas inactivadas seleccionadas en base a previos estudios de caracterizaciónde las mismas. El fenol se utilizó como inactivante mientras que como adyuvante se utilizó el hidróxido dealuminio. El trabajo evaluó la respuesta immune humoral inducida en un grupo de 15 corderos Pelibuey y 8cabritos de la raza canaria majorera, los cuales recibieron 2 dosis de vacuna a los 45 y 70 días de edad, siendomonitorizados hasta los 4 meses de vida. Los títulos de anticuerpos observados por ELISA indirecto muestran títulos menores en el grupo de corderos que en el grupo de cabritos frente a ambos antígenos, siendo estasdiferencias estadísticamente significativas sólo frente a M. agalactiae

A combined vaccine against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (largecolony type) was developed using inactivated strains selected in previous characterization studies. Phenol wasused as inactivating agent and aluminium hydroxide was added as adjuvant. The present study was designedto evaluate the specific humoral immune response to these two mycoplasma species shown by lambs and goatkids. One group of 15 Pelibuey lambs and one group of 8 Canary goat kids received two injections of vaccineat the ages 45 and 70 days and were monitored until they were 120 days old. Antibody titres were determinedby indirect ELISA. Lambs showed lower antibody levels (expressed as ODs) to both antigens than goat kidsalthought significantly differences were only registered to M. agalactiae antigen (P<0.005)