Microbiological insights into respiratory infections and the opportunities for inhaled therapy

Respiratory infections represent the fourth most common cause of all deaths across age groups and countries. Treating these infections appropriately is a clear clinical priority and here we outline the types of therapy that are in current use for some of these infections. It is important that treatments are further improved and the potential of inhaled delivery to fulfil this need is considered. We describe novel methodologies that are being applied for the identification and enumeration of microorganisms in the respiratory tract, and propose that ways of improving therapy may arise from understanding better the etiology of respiratory infection and the impact of inhaled drug therapies. The potential for translational benefits for patients are also discussed.

Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections of growing goats

High prevalence of anthelmintic-resistant gastrointestinal nematodes (GIN) in goats has increased pressure to find effective, alternative non-synthetic control methods, one of which is adding forage of the high condensed tannin (CT) legume sericea lespedeza (SL; Lespedeza cuneata) to the animal's diet. Previous work has demonstrated good efficacy of dried SL (hay, pellets) against small ruminant GIN, but information is lacking on consumption of fresh SL, particularly during the late summer–autumn period in the southern USA when perennial warm-season grass pastures are often low in quality. A study was designed to determine the effects of autumn (September–November) consumption of fresh SL forage, grass pasture (predominantly bermudagrass, BG; Cynodon dactylon), or a combination of SL + BG forage by young goats [intact male Spanish kids, 9 months old (20.7 ± 1.1 kg), n = 10/treatment group] on their GIN infection status. Three forage paddocks (0.40 ha) were set up at the Fort Valley State University Agricultural Research Station (Fort Valley, GA) for an 8-week trial. The goats in each paddock were supplemented with a commercial feed pellet at 0.45 kg/head/d for the first 4 weeks of the trial, and 0.27 kg/head/d for the final 4 weeks. Forage samples taken at the start of the trial were analyzed for crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) content, and a separate set of SL samples was analyzed for CT in leaves, stems, and whole plant using the benzyl mercaptan thiolysis method. Animal weights were taken at the start and end of the trial, and fecal and blood samples were collected weekly for determination of fecal egg counts (FEC) and packed cell volume (PCV), respectively. Adult GIN was recovered from the abomasum and small intestines of all goats at the end of the experiment for counting and speciation. The CP levels were highest for SL forage, intermediate for SL + BG, and lowest for BG forage samples, while NDF and ADF values were the opposite, with highest levels in BG and lowest in SL forage samples. Sericea lespedeza leaves had more CT than stems (16.0 g vs. 3.3 g/100 g dry weight), a slightly higher percentage of PDs (98% vs. 94%, respectively) and polymers of larger mean degrees of polymerization (42 vs. 18, respectively). There were no differences in average daily gain or blood PCV between the treatment groups, but SL goats had lower FEC (P < 0.05) than the BG or SL + BG forage goats throughout most of the trial. The SL + BG goats had lower FEC than the BG forage animals by the end of the trial (week 8, P < 0.05). The SL goats had lower numbers (P < 0.05) of male Haemonchus contortus and tended to have fewer female (P < 0.10) and total (P < 0.07) H. contortus compared with the BG goats. The predominant GIN in all the goats was Trichostrongylus colubriformis (73% of total GIN). As a low-input forage with activity against pathogenic GIN (H. contortus), SL has a potential to reduce producers’ dependence upon synthetic anthelmintics and also to fill the autumn ‘window’ in good-quality fresh forages for goat grazing in the southern USA.

Interactions between nutrition and infections with Haemonchus contortus and related gastro-intestinal nematodes in small ruminants

Interactions between host nutrition and feeding behaviour are central to understanding the pathophysiological consequences of infections of the digestive tract with parasitic nematodes. The manipulation of host nutrition provides useful options to control gastrointestinal nematodes as a component of an integrated strategy. Focused mainly on the Hameonchus contortus infection model in small ruminants, this chapter (i) illustrates the relationship between quantitative (macro- and micro-nutrients) and qualitative (plant secondary metabolites) aspects of host nutrition and nematode infection, and (ii) shows how basic studies aimed at addressing some generic questions can help provide solutions, despite the considerable diversity of epidemiological situations and breeding systems.

Analysis of cryptic, systemic Botrytis infections in symptomless hosts

Botrytis species are generally considered to be aggressive, necrotrophic plant pathogens. By contrast to this general perception, however, Botrytis species could frequently be isolated from the interior of multiple tissues in apparently healthy hosts of many species. Infection frequencies reached 50% of samples or more, but were commonly less, and cryptic infections were rare or absent in some plant species. Prevalence varied substantially from year to year and from tissue to tissue, but some host species routinely had high prevalence. The same genotype was found to occur throughout a host, representing mycelial spread. B. cinerea and B. pseudocinerea are the species that most commonly occur as cryptic infections, but phylogenetically distant isolates of Botrytis were also detected, one of which does not correspond to previously described species. Sporulation and visible damage occurred only when infected tissues were stressed, or became mature or senescent. There was no evidence of cryptic infection having a deleterious effect on growth of the host, and prevalence was probably greater in plants grown in high light conditions. Isolates from cryptic infections were often capable of causing disease (to varying extents) when spore suspensions were inoculated onto their own host as well as on distinct host species, arguing against co-adaptation between cryptic isolates and their hosts. These data collectively suggest that several Botrytis species, including the most notorious pathogenic species, exist frequently in cryptic form to an extent that has thus far largely been neglected, and do not need to cause disease on healthy hosts in order to complete their life-cycles.

Seasonal patterns of viral and bacterial infections among children hospitalized with community-acquired pneumonia in a tropical region

Community-acquired pneumonia (CAP) is a common cause of morbidity among children. Evidence on seasonality, especially on the frequency of viral and bacterial causative agents is scarce; such information may be useful in an era of changing climate conditions worldwide. To analyze the frequency of distinct infections, meteorological indicators and seasons in children hospitalized for CAP in Salvador, Brazil, nasopharyngeal aspirate and blood were collected from 184 patients aged < 5 y over a 21-month period. Fourteen microbes were investigated and 144 (78%) cases had the aetiology established. Significant differences were found in air temperature between spring and summer (p = 0.02) or winter (p < 0.001), summer and fall (p = 0.007) or winter (p < 0.001), fall and winter (p = 0.002), and on precipitation between spring and fall (p = 0.01). Correlations were found between: overall viral infections and relative humidity (p = 0.006; r = 0.6) or precipitation (p = 0.03; r = 0.5), parainfluenza and precipitation (p = 0.02; r = -0.5), respiratory syncytial virus (RSV) and air temperature (p = 0.048; r = -0.4) or precipitation (p = 0.045; r = 0.4), adenovirus and precipitation (p = 0.02; r = 0.5), pneumococcus and air temperature (p = 0.04; r = -0.4), and Chlamydia trachomatis and relative humidity (p = 0.02; r = -0.5). The frequency of parainfluenza infection was highest during spring (32.1%; p = 0.005) and that of RSV infection was highest in the fall (36.4%; p < 0.001). Correlations at regular strength were found between several microbes and meteorological indicators. Parainfluenza and RSV presented marked seasonal patterns.

The role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country

We report an investigation for 16 bacteria and viruses among 184 children hospitalized with pneumonia in Salvador, Brazil. Etiology was established in 144 (78%) cases. Viral, bacterial, and mixed infections were found in 110 (60%), 77 (42%), and 52 (28%) patients, respectively. Rhinovirus (21%) and Streptococcus pneumoniae (21%) were the most common pathogens. Our results demonstrate the importance of viral and pneumococcal infections among those patients.

Natural Plasmodium infections in Brazilian wild monkeys: Reservoirs for human infections?

Four hundred and forty-eight samples of total blood from wild monkeys living in areas where human autochthonous malaria cases have been reported were screened for the presence of Plasmodium using microscopy and PCR analysis. Samples came from the following distinct ecological areas of Brazil: Atlantic forest (N = 140), semideciduous Atlantic forest (N = 257) and Cerrado (a savannah-like habitat) (N = 51). Thick and thin blood smears of each specimen were examined and Plasmodium infection was screened by multiplex polymerase chain reaction (multiplex PCR). The frequency of Plasmodium infections detected by PCR in Alouatta guariba clamitans in the Sao Paulo Atlantic forest was 11.3% or 8/71 (5.6% for Plasmodium malariae and 5.6% for Plasmodium vivax) and one specimen was positive for Plasmodium falciparum (1.4%); Callithrix sp. (N = 30) and Cebus apella (N = 39) specimens were negative by PCR tests. Microscopy analysis was negative for all specimens from the Atlantic forest. The positivity rate for Alouatta caraya from semideciduous Atlantic forest was 6.8% (16/235) in the PCR tests (5.5, 0.8 and 0.4% for P. malariae, P. falciparum and P. vivax, respectively), while C apella specimens were negative. Parasitological examination of I he samples using thick smears revealed Plasmodium sp. infections in only seven specimens, which had few parasites (3.0%). Monkeys from the Cerrado (a savannah-like habitat) (42 specimens of A. caraya, 5 of Callithrix jacchus and 4 of C. apella) were negative in both tests. The parasitological prevalence of P. vivax and P. malariae in wild monkeys from Atlantic forest and semideciduous Atlantic forest and the finding of a positive result for P.falciparum in Alouatta from both types of forest support the hypothesis that monkeys belonging to this genus could be a potential reservoir. Furthermore, these findings raise the question of the relationship between simian and autochthonous human malaria in extra-Amazonian regions. (C) 2008 Elsevier B.V. All rights reserved.

Tamoxifen as a potential antileishmanial agent: efficacy in the treatment of Leishmania braziliensis and Leishmania chagasi infections

The aim of this study was to evaluate the efficacy of tamoxifen in vivo in experimental models of cutaneous (CL) and visceral leishmaniasis (VL) caused by Leishmania braziliensis and Leishmania chagasi, respectively. Drug activity was assessed against intracellular amastigotes by treating infected macrophage cultures and evaluating the number of infected cells. In vivo efficacy of tamoxifen was tested in L. braziliensis-infected BALB/c mice and in L. chagasi-infected hamsters. Treatment with 20 mg/kg/day tamoxifen was administered for 15 days by the intraperitoneal route. Efficacy was evaluated through measurements of lesion size, parasite burden at the lesion site or liver and spleen and survival rate. Tamoxifen killed L. braziliensis and L. chagasi intracellular amastigotes with 50% inhibitory concentrations (IC(50)) of 1.9 +/- 0.2 and 2.4 +/- 0.3 mu M, respectively. Treatment of L. braziliensis-infected mice with tamoxifen resulted in significant reductions in lesion size and 99% decrease in parasite burden, compared with mock-treated controls. L. chagasi-infected hamsters treated with tamoxifen showed significant reductions in liver parasite load expressed as Leishman-Donovan units and 95% to 98% reduction in spleen parasite burden. All animals treated with tamoxifen survived while 100% of the mock-treated animals had died by 11 weeks after the interruption of treatment. Tamoxifen is effective in the treatment of CL and VL in rodent models.

Plasmodium vivax: Microsatellite analysis of multiple-clone infections

We used mixtures of genomic DNA from two genetically distinct isolates from Brazil, 42M and 312M, to investigate how accurately 12-locus microsatellite typing describes the overall genetic diversity and characterizes multilocus haplotypes in multiple-clone Plasmodium vivax infections. We found varying PCR amplification efficiencies of microsatellite alleles; for example, from the same 1:1 mixture of 42M and 312M DNA we amplified predominantly 312M-type alleles at 10 loci and 42M-type alleles at 2 loci. All microsatellite alleles were accurately scored in 1:0.5 and 1:0.25 312M:42M DNA mixtures, even when minor peak heights did not meet previously suggested criteria for minor allele detection in multiple-clone infections. Relative proportions of major and minor alleles were unaffected by multiple displacement amplification of template DNA prior to PCR-based microsatellite typing. Although microsatellite typing may detect minor alleles in clone mixtures, amplification biases may lead to inaccurate assignment of predominant haplotypes in multiple-clone P. vivax infections. (C) 2008 Elsevier Inc. All rights reserved.

Molecular characterization of Mycobacterium massiliense and Mycobacterium bolletii in isolates collected from outbreaks of infections after laparoscopic surgeries and cosmetic procedures

An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Belem, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and. rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak all of which were originally attributed to a single strain of M. abscessus.

HEMATOLOGICAL ASSESSMENT IN FOUR BRAZILIAN TELEOST FISH WITH PARASITIC INFECTIONS, COLLECTED IN FEEFISHING FROM FRANCA, São Paulo, BRAZIL

The effects of parasitic infections in condition factor, hematocrit, hemoglobin, mean corpuscular hemoglobin concentration (MCHC), and leucocytes and thrombocytes distribution in Piaractus mesopotamicus, Leporinus macrocephalus, hybrid tambacu (P. mesopotamicus x Colossoma macropomum and Brycon amazonicus collected in feefishing from Franca, São Paulo, Brazil were evaluated. Parasitized tambacu and L. macrocephalus had higher (p<0.05) condition factor than unparasitized fish. However, the contrary occurred in P. mesopotamicus and B. amazonicus. Changes in the hematocrit, hemoglobin and MCHC were not related to parasitism. Parasitic infections did not cause effect on leucocytes and thrombocytes percentage (p>0.05) of tambacu. In P. mesopotamicus parasitized by Monogenea Anacanthorus penilabiatus and dinoflagellate Piscinoodinium pillulare, increase in monocytes and decrease in thrombocytes percentage (p<0.05) were found. However, the same parasitic association in L. macrocephalus caused a decrease in lymphocytes percentage accompanied by increase in neutrophils percentage (p<0.05). In B. amazonicus, infection by Ichthyophthirius multifiliis, P. pillulare and monogeneans caused increase in neutrophils percentage.

Observations on helminth infections of free-living and captive rheas (Rhea americana) in Brazil

The present work describes helminth infection of eight free-living and 12 captive rheas (Rhea americana) from, respectively, Pantanal of Mato Grosso do Sul State, and Jaboticabal, São Paulo State, Brazil. Captive birds were young and had a high mortality rate, while free-living birds were adult and apparently healthy. Infections were evaluated by post-mortem examination of internal organs and recovery of helminths using standard parasitological procedures. Seven species of nematodes (Sicarius uncinipenis, Torquatoides crotophaga, Deletrocephalus dimidiatus, D. cesarpintoi, Paradeletrocephalus minor, Capillaria venteli and Dicheilonema rheae) and two species of cestodes (Houttuynia struthionis and Chapmania tauricolis) were identified. P. minor, which inhabits the large intestine, was the most common helminth in free-living birds (63.9%). In captive rheas, a mean parasitic load of 173 helminths per host was found. The gizzard of these birds was the most parasitized organ and S. uncinipenis was most common (92.5%). Parasitism of free-living and captive birds and associated pathology are discussed. (c) 2005 Published by Elsevier B. V.

Natural infections with filarioidea nematodes in Hydrochaerus hydrochaeris in the floodplain of Mato Grosso do Sul, Brazil

Foram coletados 570 nematódeos Filaroidea de 36 capivaras, sacrificadas entre 1989 e 1996, onze jovens menores que seis meses e 25 adultas, provenientes da região do pantanal do Mato Grosso do Sul (Paiaguás). Cento e setenta e seis (176) espécimes de Yatesia hydrochoerus foram encontrados nas fáscias dos músculos esqueléticos, principalmente da região do obliquus externus abdominis e na face interna dos membros posteriores e trezentos e noventa e quatro (394) espécimes de Cruorifilaria tuberocauda foram coletados das artérias renais (380) e das artérias pulmonares (14) dos animais.

Neospora caninum infection in birds: experimental infections in chicken and embryonated eggs

Neospora caninum causes economical impact in cattle-raising farms since it is implicated as the major cause of bovine abortions. Although infection by the parasite has been widely described in mammals, the role of birds in its life-cycle is still obscure. Therefore, this work aimed to evaluate the infection by N. caninum in different chicken models. Experimental infections were conducted in 7-day-old chicks, laying hens and embryonated eggs, where samples were analysed for parasite burden, IgG antibodies and lesions promoted. Chickens demonstrated an asymptomatic infection, although with seroconversion and systemic replication of the parasite. In laying hens, no signs of vertical transmission were observed. However, embryonated eggs inoculated by the allantoic cavity route demonstrated susceptibility to infection, with mortality rates around 50% independent of the inoculum dose. Additionally, dogs became infected after ingestion of different amounts of inoculated eggs, producing either oocysts or specific IgG antibodies. The results herein presented demonstrate that chickens may be intermediate hosts of N. caninum and that embryonated eggs could be a useful model to study the parasite's biology.

Phenotypical characterization and adhesin identification in Escherichia coli strains isolated from dogs with urinary tract infections

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)