Intracellular killing of Candida albicans by human polymorphonuclear leucocytes: comparison of three methods of assessment.

Three different methods, [3H]uridine uptake, viable count and 51Cr-release were used to assess the intracellular survival of a strain of Candida albicans, 19321, which was lethal for mice injected intravenously. Intracellular survival 1 h after ingestion ranged from 50 to 80% depending on the method employed and the detergent used to lyse the phagocytes. Inhibition of uridine uptake by detergents used to lyse the phagocytes led to difficulty in assessment of intracellular killing by this method.

Role of maternal immunity in the protection of newborn ferrets against infection with a virulent influenza virus.

Intranasal infection of newborn ferrets with a virulent strain of influenza virus invariably resulted in their deaths following virus replication to high titre in both lung and nasal turbinates (Collie et al., 1980). However, a similar challenge of newborn ferrets born to mothers immunized by infection with virulent or attenuated viruses resulted in complete protection; no virus replicated in their lungs and little or no virus was isolated from their nasal turbinates. Protection appeared to be antibody-mediated since it was sub-type-specific and milk-derived since newborn ferrets born to non-immune mothers but fostered onto immune mothers exhibited a similar level of protection to neonates born to and suckled by immune mothers.

The similar interaction of ferret alveolar macrophages with influenza virus strains of differing virulence at normal and pyrexial temperatures.

The possibility that ferret lung macrophages may be one factor operating in vivo to prevent infection of susceptible alveolar cells (as demonstrated by organ cultures) by both virulent and attenuated strains of influenza virus has been investigated. Phagocytosis of four strains of influenza virus [A/PR/8/34 (H1N1) and clone 64d (attenuated for ferrets) and clones 64c and 7a (virulent for ferrets) of the recombinant virus A/PR/8/34-A/England/939/69 (H3N2)] by ferret alveolar macrophages in vitro showed that all strains, whether virulent or attenuated, attached equally well (72 to 93%). Recoveries of virus were similar (17 to 44%) whether phagocytosis occurred at the normal temperature of the ferret (39 degrees C) or at pyrexial temperatures induced during infection (40 degrees C for A/PR/8/34 and clone 64d; 41 degrees C for clones 7a and 64c). Thus, alveolar macrophages probably contribute to the lack of alveolar infection observed in vivo.

The effects of maternal influenzal viraemia in late gestation on the conceptus of the pregnant ferret.

Pregnant ferrets were inoculated intra-cardially on day 30 of gestation with influenza virus. The animals were sacrificed on days 5 to 11 after inoculation and the products of conception including the uterus were examined virologically and histopathologically. The results indicate that the initial site of infection of the conceptus is the haemophagous organ and that spread occurs from this site to the endometrium, placental labyrinth and fetus. Lesions in the fetus are confined to the liver and respiratory tract. In the liver they may represent either a viral hepatitis or a secondary response to placental damage resulting in the stimulation of erythropoiesis. In the respiratory tract they first occur in the nasal sinuses and upper airways suggesting that infection is via the amniotic fluid rather than via the blood stream. The relevance of these findings to human pregnancy is discussed.

Distribution of viral antigen with the lower respiratory tract of ferrets infected with a virulent influenza virus: production and release of virus from corresponding organ cultures.

Using fluorescent antibody techniques, a semi-quantitative survey has been made of the distribution of influenza virus antigen in the trachea, main bronchi, and three zones (hilar, intermediate and alveolar) of all four lung lobes of ferrets following intranasal inoculation of a virulent clone (7a) of the recombinant influenza virus A/PR/8/34-A/England/939/69 (H3N2). The results confirm the indications from our previous quantitative surveys of infectious virus and histological damage in these areas, namely that infection is confined largely to airway epithelium and is rare in the alveoli. Furthermore, in the lung zones, viral antigen resided mainly in the bronchial rather than bronchiolar epithelium. In attempts to identify the reasons for lack of alveolar involvement organ cultures of alveolar tissue, from which all major airways had been removed, produced levels of virus similar to cultures of bronchus and trachea and the hilar and intermediate lung zones which contain airway and alveolar tissue. Hence, the lack of alveolar infection in vivo must be due to factors which prevent virus attack of susceptible alveolar cells. However, these organ culture experiments showed that a contributing factor could be very poor release of virus from any alveolar cells that do become infected. In contrast, although cultures of bronchi produced less virus than those of nasal turbinates (the most susceptible tissue in vivo) they released a high proportion of their yield and this ease of release may contribute to spread of infection in vivo.

Differential replication of attenuated and virulent influenza viruses in organ cultures of ferret bronchial epithelium. Brief report.

In contrast to its abundant replication in ferret nasal epithelium in vivo and in vitro, comparable to that of the virulent strains, the attenuated influenza virus A/PR/8/34 produced much lower yields than the virulent strains in organ cultures of bronchial epithelium agreeing with its relative inability to infect the lower respiratory tract of ferrets. The replication of another attenuated strain showed different temperature characteristics in bronchial epithelium to that in nasal turbinate epithelium.

The role of cellular susceptibility in the declining severity of respiratory influenza of ferrets with age.

A comparison was made, both in vivo and in organ culture, between newborn (1-day-old) and suckling (15-day-old) ferrets of lower respiratory tract tissue infected with a virulent strain (clone 7a) of influenza virus. Newborn ferrets were killed by influenza virus following intranasal inoculation but suckling ferrets were almost as resistant as adult ferrets. In newborn ferrets there was a rapid, severe and progressive infection of lung tissue with infection of alveolar cells as well as those of bronchial and bronchiolar epithelium (assessed by monitoring virus infectivity and by fluorescent antibody staining). In suckling ferrets, as previously shown for adult animals, the lung infection was less severe, less persistent and confined to the epithelium of bronchi with only a small bronchiolar involvement and even less alveolar cell infection. These differences observed in vivo were repeated in organ cultures obtained from various areas of the lung. i.e. alveolar and airway epithelial cells of newborn ferrets exhibited a greater susceptibility than those of older ferrets. Thus, it appears that one factor determining the greater susceptibility of the lower respiratory tract of newborn ferrets is a greater inherent susceptibility of alveolar and airway epithelial cells to infection with influenza virus. Other factors may also be involved and have yet to be investigated.

The role of lung development in the age-related susceptibility of ferrets to influenza virus.

Newborn (I-day-old) ferrets died following intranasal inoculation of influenza virus (clone 7a) but suckling (15-day-old) ferrets were almost as resistant as adult ferrets. Many of the deaths in newborn ferrets were consequent upon an increased lower respiratory tract infection. One reason for the latter was an increase in susceptibility of both ciliated epithelium and alveolar cells in newborn ferret lungs when compared with the corresponding cells in adult and suckling ferrets (Coates et al. 1984). Work reported here shows that the lungs of newborn ferrets possess a greater proportion of ciliated epithelium-lined airway in comparison with the lungs of suckling and adult ferrets. This situation might also contribute to the increased susceptibility of the lower respiratory tract although the difficulties of assessing this influence precisely are discussed. In addition, the occlusion of the narrower airways of the immature lung in the infected newborn ferret contributes to the increased respiratory complications.

The relation of interferon and nonspecific inhibitors to virus levels in nasal washes of ferrets infected with influenza viruses of differing virulence.

Two clones (7a, virulent; 64d, attenuated) of a recombinant influenza virus (A/PR/8/34-A/ENGLAND/939/69 (H3N2)) were inactivated at the same rate by viral inhibitors present in nasal washes taken from both Clone 7a- and Clone 64d-infected ferrets. Both clones induced similar levels of interferon in the nasal washes of infected animals. The onset and rise of interferon production occurred at the same time for both clones, and was associated with a decline in virus titres. In addition, both clones showed a similar sensitivity to interferon. Thus, although nonspecific inhibitors and interferon may play a role in reducing nasal tract infection caused by both clones, the differences in virulence of the 2 clones do not appear to be determined by differential induction of, or resistance to, these host defence mechanisms.

Elevation of nasal viral levels by suppression of fever in ferrets infected with influenza viruses of differing virulence.

The effect of suppression of fever on viral levels in nasal washes of ferrets infected with either of two clones (7a, virulent; 64d, attenuated) of the recombinant influenza virus A/Puerto Rico/8/34-A/England/939/69 (H3N2) was studied. The febrile response was reduced by shaving the ferrets or by treating them with sodium salicylate, which had no noticeable effect on the inflammatory response. For both clones, significantly more virus was shed in the nasal washes of ferrets whose febrile response was suppressed, and the viral levels decreased less rapidly than in untreated ferrets or in those in which the treatments were ineffective.

The role of naturally-acquired bacterial infection in influenza-related death in neonatal ferrets.

Concomitant, naturally-acquired bacterial infection was the cause of some deaths occurring in neonatal ferrets infected with the attenuated influenza virus A/Puerto Rico/8/34, these being prevented by antibiotic therapy. Bacterial infection played an insignificant role in the greater number of deaths following inoculation with the virulent clone 7a (of the recombinant influenza virus A/Puerto Rico/8/34-A/England/939/69/(H3N2]. As seen previously with clone 7a some ferret neonates infected with A/PR/8/34 died either from obstruction in the upper respiratory tract or from viral pneumonia, but with the latter virus, both types of lesion were probably attributable to the bacterial superinfection.

Differential distribution of virus and histological damage in the lower respiratory tract of ferrets infected with influenza viruses of differing virulence.

The distribution of four strains of influenza virus [A/PR/8/34 (H0N1) and clone 64d (attenuated for ferrets) and clones 64c and 7a (virulent for ferrets) of the recombinant virus A/PR/8/34--A/England/939/69 (H3N2)] in the lower respiratory tract (trachea, bronchi and the hilar, intermediate and outer alveolar zones of the lung) of ferrets was monitored daily for 4 days after intranasal inoculation. On day 1, some animals had high virus titres in all the tissues but in other animals virus was undetectable, irrespective of the virus strain. Two days after inoculation increase of virus contents of all tissues tended to be restricted. On days 3 and 4, the virulent clones (64c and 7a), in contrast to the attenuated strains (A/PR/8/34 and clone 64d), consistently infected the lower respiratory tissues. However, for all infected animals the virus contents of the hilar zones of the lungs were higher than those in the intermediate zones, while the alveolar zones were relatively free from virus. Quantitative estimations of the mild histological damage occurring in the lower respiratory tract 3 to 6 days after inoculation also indicated that bronchial and bronchiolar tissue were more susceptible to influenza virus than alveolar tissue and that clones 64c and 7a produced more damage than the other two strains. In agreement with the relative viral contents of clones 64c and 7a in the bronchi and in the hilar and intermediate zones of the lung, clone 64c produced more damage than clone 7a in the bronchi and less in the bronchioles of the lung parenchyma.