Br. J. exp. Path. (1976) 57, 443
ORGAN CULTURE STUDIES ON THE EFFICIENCY OF INFECTION OF CHICKEN TISSUES WITH AVIAN INFECTIOUS BRONCHITIS VIRUS J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS From the Department of Microbiology, Houghton Poultry Re8earch Station, Houghton, Huntingdon, Cambridge PE17 2DA Received for publication March 15. 1976
Summary.-Long-term organ cultures of a range of tissues collected from specific pathogen-free chickens were employed to determine their susceptibility, and their capacity for subsequent virus production, following inoculation with avian infectious bronchitis (AIB) virus. When inoculated with approximately 2-0 log1o median ciliostatic doses (CD50) of a classical highly egg-adapted vaccine strain (H120) of AIB virus, 9 of 23 tissues were shown to be susceptible, namely the nasal turbinates, trachea, air sac membranes, lungs, proventriculus mucosa, thyroid, kidney, ovary and oviduct. When the remaining 14 tissues were inoculated with a high dose of virus (6.8 log,OCD50), the conjunctiva, caecal tonsil, testis and bursa of Fabricius were susceptible whereas the oesophagus and cloaca responded minimally. Inoculation of the same range of tissues with a high or low dose of a field strain (HVI-9) of AIB virus produced similar results, except for a number of individual variations in response, due possibly to strain differences in pathogenicity. Determinations of the minimal infectious dose requirements of the susceptible tissues revealed that the efficiency of infection with the H120 strain was highest for the nasal turbinate and tracheal tissues, and thereafter, in order of decreasing efficiency, were the air sac membranes, lung, oviduct, proventriculus mucosa, conjunctiva, kidney, ovary, bursa of Fabricius, thyroid, testis, caecal tonsil, cloaca and oesophagus. The relevance of these results is discussed in connection with the early events in the pathogenesis and the clinical syndrome of AIB infection in chickens.
AVIAN infectious bronchitis (AIB) virus is responsible for clinical respiratory disease in chickens (Beach and Schalm, 1936) but it may also cause an interstitial nephritis (Siller and Cumming, 1974). In laying hens, it can be associated with adverse effects upon egg production and quality (Delaplane and Stewart, 1939; Broadfoot and Smith, 1954; Sevoian and Levine 1957; McDougall, 1968). When AIB virus is introduced into the respiratory tract of susceptible chickens, it replicates principally in that location; and, during the initial period of infection, a viraemia can be demonstrated and other organs become infected (Kawakubo, Miyamoto and Nakamura, 1961; Hofstad and Yoder, 1966; Doherty, 1967a, 1967b; Cook, 1968). The main tropism of the
virus is not always directed towards the respiratory tract. Some strains of AIB virus, isolated originally from cases of clinical renal disease in chickens, can be re-isolated from chickens infected with such strains experimentally with a greater facility from the kidneys than from other organs (Cumming, 1969). Organ cultures of ciliated epithelium, usually in the form of tracheal explants, are increasingly employed to examine the relationship of a number of viruses with their hosts (Hoorn and Tyrrell, 1969). Cultures of this type have also been used in making primary isolations of various viral pathogens, including coronaviruses, from the respiratory tract of man (Tyrrel and Bynoe, 1965, 1966; Hoorn, 1966; Higgins, 1966).
444
J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS
(0.1 ml), allowed to adsorb while stationary for 60 min at 370, overlaid with a maintenance fluid (0.5 ml) consisting of Eagle's minimal essential medium (MEM) containing antibiotics (penicillin 100 i.u./ml and streptomycin 100 ,ug/ml), and then rolled (8 revolutions/h) at 37°. The endpoints were based on the occurrence of complete ciliostasis as a measure of the quantal response, and the results, calculated according to the method of Reed and Muench (1938), were expressed as the number of median ciliostatic doses (CD50)/ml. Chickens.-Chickens were obtained from a specific pathogen-free (SPF) flock at 2-4 weeks of age and a variety of tissues removed with aseptic precautions to supply explants for the organ culture experiments. Susceptibility of tissues.-To determine their relative susceptibility to AIB virus infection, selected tissues were removed from groups of 5 SPF chickens and cut into approximately 4-mm cubes. Hollow organs such as trachea and intestine were first opened longitudinally and cut into squares of about 4 mm. All tissues were washed once in phosphate-buffered saline (PBS), pH = 7-1, containing ten-fold the usual concentration of the antibiotics used in the 1976). Eagle's MEM. To reduce the effects of indiviAs part of a study of the early events dual variation, 2 pieces of each tissue type were bird, making 10 replicates for in the pathogenesis of AIB virus infection selected from eachtissue were individually The each type. in chickens, the investigations described inoculated (0-2 ml) withpieces virus suspension which in the present work have employed long- was allowed to adsorb in stationary tubes for term organ cultures to follow the course of 60 min at 37°. The fluid was then removed, virus growth in chicken tissue explants each tissue washed 3 times with warmed PBS to unadsorbed virus and finally overlaid which have been prepared from a variety remove with Eagle's MEM (0 5 ml). At 24-h intervals of organs. These investigations have up to 96 h, the fluids of each particular tissue sought to obtain information concerning were harvested, pooled and assayed. The the comparative efficiency of AIB virus medium was replaced and the tubes reControls, in the form of virus with infection for different organs by demon- incubated. harvested on the same medium alone, strating possible differences in suscepti- occasions. The were tubes were maintained rolling bility and by determining the minimal at 370 throughout the experiments.
It has been adequately demonstrated that chicken tracheal organ cultures are useful for the propagation of the type species member of the coronavirus group, AIB virus, after it has been adapted, first by serial passage to embryonated chicken eggs, and perhaps subsequently to tissue culture cells (Colwell and Lukert, 1969; Johnson, Newman and Wills, 1969; Cherry and Taylor-Robinson 1970; Johnson and Newman, 1971; Butler, Ellaway and Hall, 1972). It has now been shown that AIB virus will replicate in chicken tracheal organ cultures in accordance with a single cycle growth curve (Darbyshire, Cook and Peters, 1975). It is possible to make isolations of AIB virus from pathological material from infected birds directly in chick tracheal organ cultures and this technique has been shown to be preferable to the conventional use of embryonated chicken eggs (Cook, Darbyshire and Peters,
median infectious dose (MID50) requirement of tissues in organ cultures. MATERIALS AND METHODS
Virus.-The viruses used were the eggadapted H 120 vaccine strain (Massachusetts type; Noblis Poultry Products) of AIB virus, produced as described previously (Darbyshire et al., 1975); and the HVI-9 strain as described by McDougall (1968) but at the 5th passage level in embryonated eggs. Infectivity assays.-Assays of virus infectivity of samples were carried out in chick embryo tracheal organ cultures, the production and use of which have been described (Darbyshire et al., 1975; Cook et al., 1976). Four organ cultures were infected with each virus dilution
Minimal infectious dose requirements.-The MID50 requirement of AIB virus for the initiation of infection of individual tissues was calculated from determinations of their susceptibility to different virus in-puts by quantal assay after the method of Toms, Rosztoczy and Smith (1974). Ten-fold dilutions of virus were prepared and each dilution used to infect 8 replicate explants of the selected tissues, 1 explant obtained from each of 8 SPF chickens. After adsorption for 60 min at 37°, washing, and overlaying with maintenance medium as above, the cultures were re-incubated rolling at 37° for 24 h, when the medium was renewed. Assays of infectivity were made of the pooled fluid harvests of pairs of replicates after a further 24 h and the end points were calculated. When a titre of > 2-3 loglo CD50 per ml of AIB virus was
445
INFECTION OF CHICKEN TISSUES WITH AIB VIRUS
demonstrable in the pooled fluid harvests of any pair of replicates, this was considered to indicate that significant infection of those tissue explants had occurred and was accordingly scored as being positive. Over the series of virus dilutions and using the method of Reed and Muench (1938), the value of the MID50 was calculated by subtracting the titre obtained for each tissue from the original virus titre, and expressed in terms of logl0 CD50 of AIB virus. Histology.-To monitor the ability of the tissue explants to survive the conditions under which they were maintained in culture, representative portions of each were taken at the time of their collection from the chickens. Each portion was fixed in neutral buffered formalin, embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Also, at the conclusion of each experiment, all the explants were fixed and similarly treated following the collection of the fluid harvest. Uninfected tissues were also included, harvested at 24-h intervals up to 96 h and subjected to the usual fixation and staining procedures. A histological examination was made of all sections to make comparisons of the architecture of the original uninfected tissues with that of explants infected for different lengths of time.
RESULTS
Susceptibility In an initial survey of susceptibility, a series of tissues was selected for study and explants inoculated with the H120 strain of AIB virus, using a low in-put (2-0 log1OCD50) for each. In this, as in all subsequent experiments, the criterion adopted to determine the susceptibility of a particular tissue to any dose of virus was the subsequent ability of each explant to release 3 2-0 log10CD50 infectious virus into the medium (i.e. a total of 3 2-3 log1OCD50/ml of medium) at any time during the 96 h after inoculation. A further requisite was the concomitant decline in virus titre in the controls. As the result of inoculating the explants with the 2-0 log10CD50 dose, 9 tissues supported virus replication to a significant degree, thereby fulfilling the criteria for being susceptible. The remaining tissues appeared not to be susceptible as no virus production could be demonstrated. A complete list of the tissues examined and an interpretation of their response to
TABLE I.-Susceptibility of Chicken Organ Cultures to High or Low Inocula of the H120 strain of AIB virus Inoculum
Organ culture Conjunctiva Nasal turbinates
(A+ mucosa)
Trachea Air sac membrane (anterior and posterior thoracic) Lung Oesophagus Proventriculus mucosa Gizzard mucosa Duodenum Large intestine Caecal tonsil Caecum
20 log,0CD60logr 68 CD50 +
NT
+ +
NT NT
NT
NT ±
Cloaca NT Thyroid Liver Spleen Cardiac muscle NT Kidney Pancreas NT + Ovary NT Oviduct Testis + Bursa of Fabricius Results of 4 experiments. In the initial experiment all tissues were inoculated with the low dose of virus; in subsequent experiments the susceptible tissues were inoculated with the low dose, but the remainder were given the high dose. +, susceptible; i , doubtful susceptibility; -, not susceptible;
NT, not tested.
infection with 2-0 log1OCD50 of H120 strain is given in Table I. In a series of further experiments, the same range of tissues was inoculated with the H120 strain, but on these occasions 2 different doses were employed. Those tissues which were considered to be susceptible, as judged by the results of the first experiment, were inoculated with 2-0 log,OCD50 of virus as before. The tissues which had previously been designated as not susceptible were inoculated with a high dose of virus having a mean level over the series of 6-8 log,OCD50. As before, the fluids of the 10 tissue replicates of each type were pooled at 24-h intervals and assayed for the amounts of virus released each 24 h during the 96 h following inoculation. The results of 3
J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS
446
6-
4-
24
Nasal Turbinate
Trachea
0
Air Sac
1~~~~~~~
I
1
6-7 4i
cn
0
2-
Proventriculus
Lung o
I
-
l
Thyroid l
6-
44
2-
Kidney 0
24 48 72 96
Oviduct
Ovary 0
24
48
72
96
0
24
48
72
96
Hours FiG. 1. Daily release of virus into the maintenance medium (log10CD50/ml) of organ cultures of susceptible tissues inoculated with 2 -0 logjOCD50 of AIB virus (strain H120).
replicate experiments confirmed the original findings that 9 tissues were susceptible (see Table I) and the mean plots of their virus production are illustrated in Fig. 1. The upper respiratory tract tissues (nasal turbinates, trachea) responded the most rapidly, reaching maximum titres in the initial 24 h, whereas the lower tract tissues (air sac membranes, lung) attained comparable maximum virus production but within 48 to 72 h (see Fig. 1). Tissues originating from the urogenital tract were also
susceptible, including the kidney and oviduct which permitted virus replication comparable in timing and titre to the tissues of the lower respiratory system. The ovary produced virus only minimally in response to the low virus dose. The thyroid proved more efficient, however, and produced virus steadily throughout the period of study. A finding of note was that the mucosa of the proventriculus (or glandular stomach) could sustain virus replication to a significant degree in the same period.
INFECTION OF CHICKEN TISSUES WITH AIB VIRUS
447
j
Bursa
Conjunctiva , ,
-
[ I
--I~~~~~~~~~~~~
0 to
0
oa
0
6-
4-
2-
Oesophagus 0
24
48
72
96
24
48
78
96
Hours
FIG. 2.-Daily release of virus into the maintenance medium (log,OCD50/ml) of organ cultures of tissues inoculated with 6-2 log,OCD50 of AIB virus (strain H120). In an initial experiment, these types of tissue did not respond to inoculation with 2 0 log1OCD50 of the same virus.
When those tissues originally considered not to be susceptible were inoculated with a high dose of virus, the conjunctiva, caecal tonsil, testis and bursa of Fabricius each responded by yielding significant amounts of virus; the oesophagus and cloaca responded only minimally, and the remainder failed to produce virus at all (Table I). The results of assays of the virus production of these 6 tissues are illustrated in Fig. 2. In view of the aforegoing results further studies were made of the effect of 3 different virus in-puts in a limited range of susceptible tissues. Three groups of 10 replicates of each explant were infected with the H120 strain of AIB virus (0-2 ml) in the usual manner, the explants
of each group being inoculated with 7'0 log1oCD50, 44) log1OCD50 or 240 log10 CD50 respectively. The mean assay results for at least 2 replicate experiments of infecting 4 of the more susceptible tissues with 3 doses of virus are presented in Fig. 3, and those from similarly infecting 3 less susceptible tissues are given in Fig. 4. The lung, kidney and oviduct yielded comparable amounts of virus by 96 h irrespective of the level of in-put, whereas the proventriculus only responded when it received the high and medium doses. The testis, bursa of Fabricius and oesophagus supported virus replication following a high in-put in a manner analogous to the previous experiments
4483
J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS -
Kidney i 0
n
0
High
A-
cJ 0 C"
0
8
-
A
A 4
2
11
A
Oviduct 24
48
72
0
96
24
48
72
96
Hours FIG. 3.-Daily release of virus into the maintenance medium (log1OCD50/ml) of susceptible tissues inoculated with 3 in-put levels of AIB virus (strain H120).
(see Fig. 2), but in each case the medium dose produced a minimal response and the low dose produced no significant amounts of virus (Fig. 4). To test the possibility that differences in tissue susceptibility could vary according to the virulence of the strain of AIB virus, the HVI-9 strain was used to infect a series of tissue explants in a manner similar to the previous experiments. Based on the results obtained with the H120 strain, most tissues were inoculated
organ
cultures of 4
with either a low dose (2.0 log1OCD50) or a high dose (6.3 log1OCD50) of virus with the exception of 6 tissues which were inoculated with both the high and low doses. The results are given in Table II. The respiratory tract tissues and the oviduct were again susceptible to a low virus in-put, while the conjunctiva also responded to a low dose of this strain. The proventriculus mucosa, thyroid, kidney and ovary produced virus only in response to a high dose of the HVI-9
449
INFECTION OF CHICKEN TISSUES WITH AIB VIRUS
8Bursa
Oesophagus
6-
0
4-
von
Ca0 2
0
24
48
72
96
24
0
48
72
96
0
24
48
72
96
Hours FIG. 4. Daily release of virus into the maintenance medium (log,0CD50/ml) of organ cultures of 3 tissues, not originally designated as susceptible, inoculated with 3 in-put levels of AIB virus (strain H120).
strain. As with the H120 strain, the testis, bursa of Fabricius, caecal tonsil and cloaca were susceptible to a high dose. The tissues which were not susceptible to a high in-put of the H120 strain, with the
addition of the oesophagus, were not susceptible to a high dose of the HVI-9 strain.
Minimal infectious dose determinations The range of tissues selected for the TABLE II.-Susceptibility of Chicken Organ Cultures to High or Low Inocula of the determination of the MID50 was restricted to those susceptible to the H120 strain in HVI-9 strain of AIB virus the previous experiments. The MID50 Inoculum determinations were calculated from the 2-0 log10CD50 63 logCD50 Organ cultures results of assays of cultures 48 h after Conjunctiva + inoculation, the medium being changed Nasal turbinates NT + after the first 24 h. The period between Trachea NT + Air sac membranes NT + 24 and 48 h was considered to be the point Lung on the basis of monitoring virus when, NT Oesophagus Proventriculus mucosa decay in the controls, the presence of Gizzard mucosa NT unadsorbed residual virus could be disDuodenum NT counted. The mean results of at least 2 NT Large intestine _ Caecal tonsil NT replicate experiments are given in Table Caecum NT III. This list of tissues is arranged in ~+ Thyroid order of increasing MID50 requirements Liver NT NT Spleen for the initiation of a significant infection Cardiac muscle NT with the H120 strain of AIB virus. Kidney The tissues originating from the Pancreas Ovary respiratory system possessed in general the Oviduct NT + lowest MID50 requirements and that of the Testis NT + NT Bursa of Fabricius + nasal turbinates was comparable with the Cloaca trachea which in the present work was also Results of at least 2 experiments: +, susceptible; used as the standard assay system. The not susceptible; ±, doubtful susceptibility; air sac membranes had a somewhat NT, not tested. +
NT
-±
+
NT_
+
-,
450
J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS
TABLE III.-Minimal Infectious Dose although the visceral organs such as the (MID50) Requirement of Susceptible liver, pancreas and kidney showed discrete Chicken Tissues in Organ Culture using evidence of cloudy swelling and early the H120 strain of AIB virus degenerative necrosis in the centre of the explant. MID50 Tissue (logl0 CD50) In the infected cultures, over the same Nasal turbinates 0 3 the histological findings were little period, Trachea 0 3 different, with the principal exception of Air sac membranes 1*4 Lung 2 -2 the trachea. In the latter there was Oviduct 2 -2 progressive damage to the superficial Proventriculus mucosa 2-7 epithelial cells of the lumen accompanied Conjunctiva Kidney 3 9 by exfoliation of the ciliated cell layer, an 4-6 Ovary accumulation of cell debris in the lumen Bursa of Fabricius and a general thinning of the epithelial Thyroid 4*8 Testis 4.9 lining. A very limited cellular migration Caecal tonsil 5.9 and a number of pyknotic nuclei were both 5-9 Cloaca Oesophagus 6-1 apparent, although the basal cell layer Mean results of at least 2 replicate experiments. remained intact. The deeper layers of the Mean virus in-put 6 6 logl0 CD 50/0 * 2 ml. tracheal ring, including the cartilaginous tissue, remained unaltered. the intestinal tissues, the mucosa higher MID50 value, but this in turn was wasInundamaged and its structure was well lower than that of the lung, which had the preserved. The epithelial linings were highest value of any respiratory tissue. intact throughout and no lesions were The oviduct possessed a comparable evident in the mucosa of the provenMID50 value to the lung, and the proven- triculus. triculus mucosa could be infected successIn the case of the other infected fully with an average of only 0 5 log10 CD50 organs, the liver showed incipient necrosis more virus. The conjunctiva can be said of the central area, extending to about to be associated with the respiratory tract one-third of the explant within 48 h and by way of the nasal cavity, but, in contrast to approximately two-thirds by 96 h, to the true respiratory tract tissues, its with a rim of apparently healthy tissue MID50 requirement was distinctly higher surrounding it. A similar process, but to than any of them, and comparable with a lesser extent, was noted in the kidney, that of the kidney. A group of tissues pancreas, spleen and lung. In each case, comprising the ovary, bursa of Fabricius, no specific pathological changes could be thyroid and testis all had a similar facility confidently ascribed to the results of AIB for infection by the H120 strain, whereas virus infection. Furthermore, no evithe caecal tonsil, cloaca and oesophagus dence was observed of any cellular had significantly increased MID50 figures, dedifferentiation in any of the tissue the latter possessing the highest for all the explants. tissues under investigation. 3-7
4.7
DISCUSSION
Histological examinations In the haematoxylin and eosin-stained sections of the explants which were left uninoculated for a period of 96 h, the histological architecture was generally not visibly affected and was apparently well maintained. Hollow organs such as the trachea and intestine showed no change,
The technique of using roller tubes for the culture of chicken tracheal explants is an established practice (Harnett and Hooper, 1968; Cherry and TaylorRobinson, 1970; Butler et al., 1972; Darbyshire et al., 1975; Cook et al., 1976) and in the present studies was extended to the maintenance of explants from other
INFECTION OF CHICKEN TISSUES WITH AIB VIRUS
tissues, as it was considered preferable to the conventional use of Petri dishes as described by Hoorn (1964). The explants were bathed regularly by the maintenance fluid during rolling which resulted in the retention of an adequate cellular architecture within infected or uninfected tissues and in satisfactory virus production. The apparent absence of histological changes directly attributable to AIB virus infection implies that specific viral damage of cells was minimal. It is possible that, despite their paucity, any specific cytopathological changes would be obscured by the natural degenerative processes occurring in the explants as culture continued. The H120 strain is widely employed as a live vaccine in disease control, and provides a convenient model for the study of the pathogenesis of AIB virus. The results obtained here from a survey of the susceptibility of a range of chicken organs indicate that tissues of the respiratory system, particularly those of the upper part of the tract, are more susceptible to AIB virus infection than those from other sites. This is not unexpected in view of the findings of a number of workers who have studied the experimental disease in chickens. Kawakubo et al. (1961) infected chickens with the CAV strain of AIB virus by various routes and recovered it in highest titre in the tracheal mucus and from the lungs. Virus was also found in intestinal contents for up to 21 days after infection but active infection of any specific site was not described. Hofstad and Yoder (1966) administered a number of strains to chickens by aerosol and subsequently recovered them in greatest amounts from the trachea, lung and air sacs, although virus was also present in lower titres in the kidney, pancreas, spleen, liver and the bursa of Fabricius. Doherty (1967b) assayed virus from tissues of infected chickens and recovered it for up to 7 days, mostly from the lungs and trachea, as well as from the kidney, spleen, bursa of Fabricius, proventriculus, liver and heart.
451
In each of the aforegoing investigations (Kawakubo et al., 1961; Hofstad and Yoder, 1966; Doherty, 1967b) a viraemia was demonstrated, although there is a lack of agreement as to its timing or duration. The collection of tissues which may contain infective blood could lead to an apparent demonstration of virus in otherwise insusceptible tissues. In this respect the findings with the infection of explants in vitro may reflect more accurately the early events in the pathogenesis of AIB virus infection in chickens. In interpreting the results from the present work it is assumed that a lack of susceptibility of tissues in culture reflects their insusceptibility in vivo. In a brief report, Jones (1973) has stated that when the M41 (Massachusetts) strain of AIB virus was used to infect a limited number of chicken organ cultures, it replicated well in the trachea and oviduct, but only at a low level in lung, kidney, spleen and small intestine. When another chicken was used only the trachea responded to infection and this was ascribed to individual variation. In the present studies, an attempt has been made to obviate such variation by replicate sampling involving numbers of chickens of a similar age. The MID50 values provide a quantitative and relative assessment of the efficiency with which AIB virus would initiate the infection of various tissues in the susceptible chicken. These values correlate with the results of the surveys for tissue susceptibility and confirm that the upper part of the respiratory tract was the most vulnerable and more so than the air sac membranes, followed in turn by the lungs and oviduct which were themselves both comparable. The oesophagus was the least easily infected (see Fig. 2 and 4). In most respects the results of infecting tissues with the HVI-9 strain were similar to those obtained with the H120 strain, when used at 2 levels of in-put, with the exception of the thyroid, kidney and ovary, due possibly to differences in strain pathogenicity (Table II). The isolation
452
J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS
of AIB virus from the kidneys of chickens infected experimentally has been reported frequently (Kawakubo et al., 1961; Hofstad and Yoder, 1966; Doherty 1967b; Cumming 1969), although certain strains have a definitive tropism for that organ in field cases (Cumming, 1962, 1963, 1969). The significance of strain as well as serotype differences in artificial infections of organ cultures is accordingly now under further investigation. The susceptibility of the oviduct to both strains (H120, HVI-9), together with its comparability in virus production with tissues from the lower respiratory tract, reflects its involvement in the natural disease. Experimental infection of dayold chicks or laying hens can lead to abnormalities of the reproductive tract and variations in egg quality (Crinion, Ball and Hofstad, 1971a; Jones and Jordan, 1972; McDougall, 1968), for the virus infects the epithelial cells of the oviduct of young chicks or older birds, probably as the result of a viraemia, and may be recovered from that site for up to 11 days after experimental infection (Crinion et al., 1971b; Jones and Jordan, 1971). The susceptibility of the proventriculus mucosa accords with the recovery of virus from that site in chickens infected experimentally by Doherty (1967b) who considered that it may have been associated with virus in ingested tracheal mucus. The ability of the mucosa to be infected in culture suggests that it could acquire infection in vivo either by ingestion or as the result of a viraemia. The definitive reaction of the cloaca to a high in-put of the HVI-9 strain and the lesser one to the H120 strain implies that each could initiate infection of that organ. This supports the findings of Hudson and Beaudette (1933) that birds can be infected experimentally via the cloaca, so that this organ must be regarded as a potential portal of infection in vivo. The more susceptible tissues, such as lung, oviduct and kidney, each produced comparable virus yields by 96 h when
different inocula were used (see Fig. 3), and there may be a similar course of events in vivo with such tissues whatever the level of infection. This was also the case, in part, with the proventriculus mucosa, but its poor response in this series of experiments to the low dose of virus in view of the previous findings (see Fig. 1) needs further investigation. The responses of the less susceptible tissues, like testis, bursa of Fabricius and oesophagus, indicate that, with the H120 strain at least, a threshold level of virus is required before these tissues will respond to infection (Fig. 4). The establishment of the infection of organs with AIB virus in natural infections in vivo depends on a number of factors, including numbers and availability of susceptible cells as well as differences in pathogenicity between virus strains. The respiratory tract is the obvious target organ, the present results showing that it is the easiest to infect and, in the upper part, produces virus the most rapidly. This provides epidemiological supportive evidence for the rapid spread of virus within a population of susceptible birds. In this respect, a primary infection of the conjunctiva per se would appear to play a less significant role. If the respiratory tract were not infected initially, then alternatively various parts of the alimentary tract could permit virus production, including the proventriculus mucosa. The innate resistance of other parts of the alimentary tract must be due to an unavailability of susceptible cells, in view of the insusceptibility of washed explants, rather than to adverse chemical and pH conditions or the presence of deleterious enzymes. The importance of local production of AIB virus in the alimentary tract is exemplified by the finding by Pette (1959) that virus could be recovered from the cloacal contents for up to 24 days following oral infection. The infection of certain organs in vivo would rely on the appearance of circulating virus. In low titres the latter would presumably infect those tissues considered susceptible
INFECTION OF CHICKEN TISSUES WITH AIB VIRUS
to a low virus in-put in this work, whereas circulating virus in high titre would further infect such tissues as the testis, bursa of Fabricius and oesophagus. Other factors in the pathogenesis of AIB virus infection in vivo concern the availability of cells for virus adsorption and penetration, and the circumvention of such local barriers as antibody and cellular mechanisms. Organ cultures provide models of less complexity than do experimentally infected chickens, since local antibody production and cellular defence activities are probably minimal or in abeyance. The infection of organ cultures differs, moreover, from that of tissue culture monolayers in so far as the cells in an explant are fully differentiated and would possess a range of varying susceptibility. The role of interferon and the effects of its possible differential production by the various cell types are also aspects for further consideration. In the present investigations the use of organ cultures to examine and quantify the efficiency of AIB virus infection of chicken tissues has now been established. REFERENCES BEACH, J. R. & SCHALM, 0. W. (1936) A Filterable Virus Distinct from that of Laryngotracheitis, the Cause of a Respiratory Disease of Chicks. Poultry Sci., 15, 199. BROADFOOT, D. I. & SMITH, W. M. (1954) Effects of Infectious Bronchitis in Laying Hens on Egg Production, per cent Unsettable Eggs and Hatchability. Poultry Sci., 33, 653. BUTLER, M., ELLAWAY, W. J. & HAALL, T. (1972) Comparative Studies on the Infectivity of Avian Respiratory Viruses for Eggs, Cell Cultures and Tracheal Explants. J. comp. Path., 82, 327. CHERRY, J. D. & TAYLOR-RoBINsoN, D. (1970) Large Quantity Production of Chicken Embryo Tracheal Organ Cultures and Use in Virus and Mycoplasma Studies. Appl. Microbiol., 19, 658. COLWELL, W. M. & LUKERT, P. D. (1969) Effects of Avian Infectious Bronchitis Virus (IBV) on Tracheal Organ Cultures. Avian Di8., 13, 888. COOK, J. K. A. (1968) Duration of Experimental Infectious Bronchitis in Chickens. Re8. vet. Sci., 9, 506. COOK, J. K. A., DARBYSHIRE, J. H. & PETERS, R. W. (1976) The Use of Chicken Tracheal Organ Cultures for the Isolation and Assay of Avian Infectious Bronchitis Virus. Archiv. Virol., 50, 109. CRINION, R. A. P., BALL, R. A. & HOFSTAD, M. S. (1971a) Pathogenesis of Oviduct Lesions in Immature Chickens Following Exposure to
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Infectious Bronchitis Virus at One Day Old. Avian Di8., 15, 32. CRINION, R. A. P., BALL, R. A. & HOFSTAD, M. S. (1971b) Abnormalities in Laying Chickens Following Exposure to Infectious Bronchitis Virus at One Day Old. Avian Dis., 15, 42. CUMMING, R. B. (1962) The Aetiology of Viraemia of Chickens. Au8t. vet J., 38, 554. CUMMING, R. B. (1963) Infectious Avian Nephrosis (Uraemia) in Australia. Au8t. vet. J., 39, 145. CUMMING, R. B. (1969) Studies on Avian Infectious Bronchitis Virus. I. Distribution and Survival of the Virus in Tissues of Affected Chickens and Studies on the Carrier Status. Au8t. vet. J., 45, 305. DARBYSHIRE, J. H., COOK, J. K. A. & PETERS, R. W. (1975) Comparative Growth Kinetic Studies on Avian Infectious Bronchitis Virus in Different Systems. J. comp. Path., 85, 623. DELAPLANE, J. P. & STEWART, H. 0. (1939) Studies of Infectious Bronchitis. Bull. Rhode Ila. agric. Exp. Stn., 237. DOHERTY, P. C. (1967a) Occurrence of Avian Infectious Bronchitis Virus in the Tissues of Experimentally Infected Chickens. Qd. J. Agric. anim. Sci., 24, 75. DOHERTY, P. C. (1967b) Titration of Avian Infectious Bronchitis Virus in the Tissues of Experimentally Infected Chickens. Au8t. vet. J., 43, 575. HARNETT, G. B. & HOOPER, W. L. (1968) Test-tube Organ Cultures of Ciliated Epithelium for the Isolation of Respiratory Viruses. Lancet, i, 339. HIGGINS, P. G. (1966) The Isolation of Viruses from Acute Respiratory Infections. V. The Use of Organ Cultures of Human Embryonic Nasal and Tracheal Epithelium. Mon. Bull. Mini8t. Hlth., 25, 283. HOFSTAD, M. S. & YODER, H. W. (1966) Avian Infectious Bronchitis-Virus Distribution in Tissues of Chicks. Avian Di8., 10, 230. HOORN, B. (1964) Respiratory Viruses in Model Experiments. Transaction of the 15th Congress of the Scandinavian Oto-laryngological Society 1963. Acta oto-lar., 188, Suppl., 138. HOORN, B. (1966) Organ Cultures of Ciliated Epithelium for the Study of Respiratory Viruses. Acta path. microbiol. 8cand., 183, Suppl. 66. HOORN, B. & TYRRELL, D. A. J. (1969) Organ Cultures in Virology. Prog. med. Virol., 11, 408. HUDSON, C. B. & BEAUDETTE, F. R. (1933) The Susceptibility of Cloacal Tissue to the Virus of Infectious Bronchitis. Cornell Vet., 23, 63. JOHNSON, R. B. & NEWMAN, J. A. (1971) Measurement of Antigenic and Pathogenic Variability of Infectious Bronchitis Virus Strains by Trachea Culture. Avian Di8., 15, 233. JOHNSON, R. B., NEWMAN, J. A. & WILLS, F. K. (1969) Titration of Infectious Bronchitis Virus in Tracheas of Susceptible and Immune Chickens. Avian Di8., 13, 632. JONES, R. C. (1973) The Use of a Combined Immunofluorescence-Organ Culture System for Studying Infectious Bronchitis Virus. Proc. Wld Vet. Poult. As8oc., 2, 780. JONES, R. C. & JORDAN, F. T. W. (1971) The Site of Replication of Infectious Bronchitis Virus in the Oviduct of Experimentally Infected Hens. Vet. Rec., 89, 317. JONES, R. C. & JORDAN, F. T. W. (1972) Persistence of Virus in the Tissues and Development of the
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Oviduct in the Fowl Following Infection at Day Old with Infectious Bronchitis Virus. Re8. vet. Sci., 13,52. KAWAKUBO, A., MIYAMOTO, T. & NAKAMURA, J. (1961) Studies on Infectious Bronchitis of Chickens. III. Experiments with Artifically Infected Chickens. N.I.B.S. Bull. biol. Re8., 6, 1. MCDOUGALL, J. S. (1968) Infectious Bronchitis in Laying Fowls. Its Effect upon Egg Production and Subsequent Egg Quality. Vet. Rec., 83, 84. PETTE, J. (1959) Zur Ausscheidung des Huhnerbronchitisvirus im Kloakeninhalt. Mh. Tierheilk., 11, 296. REED, L. J. & MUENCH, H. (1938) A Simple Method of Estimating Fifty per Cent End Points. Am. J. Hyg., 27, 493. SEVOIAN, M. & LEVINE, P. P. (1957) Effects of Infectious Bronchitis on the Reproductive Tracts,
Egg Production and Egg Quality of Laying Chickens. Avian Di8., 1, 136. SILLER, W. G. & CUMMING, R. B. (1974) The Histopathology of an Interstitial Nephritis in the Fowl Produced Experimentally with Infectious Bronchitis Virus. J. Path., 114, 163. ToMs, G. L., RoSZTOCZY, I. & SMITH, H. (1974) The Localization of Influenza Virus: Minimal Infectious Dose Determinations and Single Cycle Kinetic Studies on Organ Cultures of Respiratory and Other Ferret Tissues. Br. J. exp. Path., 55, 116. TYRRELL, D. A. J. & BYNOE, M. L. (1965) Cultivation of a Novel Type of Common-Cold Virus in Organ Cultures. Br. med. J., i, 1467. TYRRELL, D. A. J. & BYNOE, M. L. (1966) Cultivation of Viruses from a High Proportion of Patients with Colds. Lancet, i, 76.
ORGAN CULTURE STUDIES ON THE EFFICIENCY OF INFECTION OF CHICKEN TISSUES WITH AVIAN INFECTIOUS BRONCHITIS VIRUS J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS From the Department of Microbiology, Houghton Poultry Re8earch Station, Houghton, Huntingdon, Cambridge PE17 2DA Received for publication March 15. 1976
Summary.-Long-term organ cultures of a range of tissues collected from specific pathogen-free chickens were employed to determine their susceptibility, and their capacity for subsequent virus production, following inoculation with avian infectious bronchitis (AIB) virus. When inoculated with approximately 2-0 log1o median ciliostatic doses (CD50) of a classical highly egg-adapted vaccine strain (H120) of AIB virus, 9 of 23 tissues were shown to be susceptible, namely the nasal turbinates, trachea, air sac membranes, lungs, proventriculus mucosa, thyroid, kidney, ovary and oviduct. When the remaining 14 tissues were inoculated with a high dose of virus (6.8 log,OCD50), the conjunctiva, caecal tonsil, testis and bursa of Fabricius were susceptible whereas the oesophagus and cloaca responded minimally. Inoculation of the same range of tissues with a high or low dose of a field strain (HVI-9) of AIB virus produced similar results, except for a number of individual variations in response, due possibly to strain differences in pathogenicity. Determinations of the minimal infectious dose requirements of the susceptible tissues revealed that the efficiency of infection with the H120 strain was highest for the nasal turbinate and tracheal tissues, and thereafter, in order of decreasing efficiency, were the air sac membranes, lung, oviduct, proventriculus mucosa, conjunctiva, kidney, ovary, bursa of Fabricius, thyroid, testis, caecal tonsil, cloaca and oesophagus. The relevance of these results is discussed in connection with the early events in the pathogenesis and the clinical syndrome of AIB infection in chickens.
AVIAN infectious bronchitis (AIB) virus is responsible for clinical respiratory disease in chickens (Beach and Schalm, 1936) but it may also cause an interstitial nephritis (Siller and Cumming, 1974). In laying hens, it can be associated with adverse effects upon egg production and quality (Delaplane and Stewart, 1939; Broadfoot and Smith, 1954; Sevoian and Levine 1957; McDougall, 1968). When AIB virus is introduced into the respiratory tract of susceptible chickens, it replicates principally in that location; and, during the initial period of infection, a viraemia can be demonstrated and other organs become infected (Kawakubo, Miyamoto and Nakamura, 1961; Hofstad and Yoder, 1966; Doherty, 1967a, 1967b; Cook, 1968). The main tropism of the
virus is not always directed towards the respiratory tract. Some strains of AIB virus, isolated originally from cases of clinical renal disease in chickens, can be re-isolated from chickens infected with such strains experimentally with a greater facility from the kidneys than from other organs (Cumming, 1969). Organ cultures of ciliated epithelium, usually in the form of tracheal explants, are increasingly employed to examine the relationship of a number of viruses with their hosts (Hoorn and Tyrrell, 1969). Cultures of this type have also been used in making primary isolations of various viral pathogens, including coronaviruses, from the respiratory tract of man (Tyrrel and Bynoe, 1965, 1966; Hoorn, 1966; Higgins, 1966).
444
J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS
(0.1 ml), allowed to adsorb while stationary for 60 min at 370, overlaid with a maintenance fluid (0.5 ml) consisting of Eagle's minimal essential medium (MEM) containing antibiotics (penicillin 100 i.u./ml and streptomycin 100 ,ug/ml), and then rolled (8 revolutions/h) at 37°. The endpoints were based on the occurrence of complete ciliostasis as a measure of the quantal response, and the results, calculated according to the method of Reed and Muench (1938), were expressed as the number of median ciliostatic doses (CD50)/ml. Chickens.-Chickens were obtained from a specific pathogen-free (SPF) flock at 2-4 weeks of age and a variety of tissues removed with aseptic precautions to supply explants for the organ culture experiments. Susceptibility of tissues.-To determine their relative susceptibility to AIB virus infection, selected tissues were removed from groups of 5 SPF chickens and cut into approximately 4-mm cubes. Hollow organs such as trachea and intestine were first opened longitudinally and cut into squares of about 4 mm. All tissues were washed once in phosphate-buffered saline (PBS), pH = 7-1, containing ten-fold the usual concentration of the antibiotics used in the 1976). Eagle's MEM. To reduce the effects of indiviAs part of a study of the early events dual variation, 2 pieces of each tissue type were bird, making 10 replicates for in the pathogenesis of AIB virus infection selected from eachtissue were individually The each type. in chickens, the investigations described inoculated (0-2 ml) withpieces virus suspension which in the present work have employed long- was allowed to adsorb in stationary tubes for term organ cultures to follow the course of 60 min at 37°. The fluid was then removed, virus growth in chicken tissue explants each tissue washed 3 times with warmed PBS to unadsorbed virus and finally overlaid which have been prepared from a variety remove with Eagle's MEM (0 5 ml). At 24-h intervals of organs. These investigations have up to 96 h, the fluids of each particular tissue sought to obtain information concerning were harvested, pooled and assayed. The the comparative efficiency of AIB virus medium was replaced and the tubes reControls, in the form of virus with infection for different organs by demon- incubated. harvested on the same medium alone, strating possible differences in suscepti- occasions. The were tubes were maintained rolling bility and by determining the minimal at 370 throughout the experiments.
It has been adequately demonstrated that chicken tracheal organ cultures are useful for the propagation of the type species member of the coronavirus group, AIB virus, after it has been adapted, first by serial passage to embryonated chicken eggs, and perhaps subsequently to tissue culture cells (Colwell and Lukert, 1969; Johnson, Newman and Wills, 1969; Cherry and Taylor-Robinson 1970; Johnson and Newman, 1971; Butler, Ellaway and Hall, 1972). It has now been shown that AIB virus will replicate in chicken tracheal organ cultures in accordance with a single cycle growth curve (Darbyshire, Cook and Peters, 1975). It is possible to make isolations of AIB virus from pathological material from infected birds directly in chick tracheal organ cultures and this technique has been shown to be preferable to the conventional use of embryonated chicken eggs (Cook, Darbyshire and Peters,
median infectious dose (MID50) requirement of tissues in organ cultures. MATERIALS AND METHODS
Virus.-The viruses used were the eggadapted H 120 vaccine strain (Massachusetts type; Noblis Poultry Products) of AIB virus, produced as described previously (Darbyshire et al., 1975); and the HVI-9 strain as described by McDougall (1968) but at the 5th passage level in embryonated eggs. Infectivity assays.-Assays of virus infectivity of samples were carried out in chick embryo tracheal organ cultures, the production and use of which have been described (Darbyshire et al., 1975; Cook et al., 1976). Four organ cultures were infected with each virus dilution
Minimal infectious dose requirements.-The MID50 requirement of AIB virus for the initiation of infection of individual tissues was calculated from determinations of their susceptibility to different virus in-puts by quantal assay after the method of Toms, Rosztoczy and Smith (1974). Ten-fold dilutions of virus were prepared and each dilution used to infect 8 replicate explants of the selected tissues, 1 explant obtained from each of 8 SPF chickens. After adsorption for 60 min at 37°, washing, and overlaying with maintenance medium as above, the cultures were re-incubated rolling at 37° for 24 h, when the medium was renewed. Assays of infectivity were made of the pooled fluid harvests of pairs of replicates after a further 24 h and the end points were calculated. When a titre of > 2-3 loglo CD50 per ml of AIB virus was
445
INFECTION OF CHICKEN TISSUES WITH AIB VIRUS
demonstrable in the pooled fluid harvests of any pair of replicates, this was considered to indicate that significant infection of those tissue explants had occurred and was accordingly scored as being positive. Over the series of virus dilutions and using the method of Reed and Muench (1938), the value of the MID50 was calculated by subtracting the titre obtained for each tissue from the original virus titre, and expressed in terms of logl0 CD50 of AIB virus. Histology.-To monitor the ability of the tissue explants to survive the conditions under which they were maintained in culture, representative portions of each were taken at the time of their collection from the chickens. Each portion was fixed in neutral buffered formalin, embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Also, at the conclusion of each experiment, all the explants were fixed and similarly treated following the collection of the fluid harvest. Uninfected tissues were also included, harvested at 24-h intervals up to 96 h and subjected to the usual fixation and staining procedures. A histological examination was made of all sections to make comparisons of the architecture of the original uninfected tissues with that of explants infected for different lengths of time.
RESULTS
Susceptibility In an initial survey of susceptibility, a series of tissues was selected for study and explants inoculated with the H120 strain of AIB virus, using a low in-put (2-0 log1OCD50) for each. In this, as in all subsequent experiments, the criterion adopted to determine the susceptibility of a particular tissue to any dose of virus was the subsequent ability of each explant to release 3 2-0 log10CD50 infectious virus into the medium (i.e. a total of 3 2-3 log1OCD50/ml of medium) at any time during the 96 h after inoculation. A further requisite was the concomitant decline in virus titre in the controls. As the result of inoculating the explants with the 2-0 log10CD50 dose, 9 tissues supported virus replication to a significant degree, thereby fulfilling the criteria for being susceptible. The remaining tissues appeared not to be susceptible as no virus production could be demonstrated. A complete list of the tissues examined and an interpretation of their response to
TABLE I.-Susceptibility of Chicken Organ Cultures to High or Low Inocula of the H120 strain of AIB virus Inoculum
Organ culture Conjunctiva Nasal turbinates
(A+ mucosa)
Trachea Air sac membrane (anterior and posterior thoracic) Lung Oesophagus Proventriculus mucosa Gizzard mucosa Duodenum Large intestine Caecal tonsil Caecum
20 log,0CD60logr 68 CD50 +
NT
+ +
NT NT
NT
NT ±
Cloaca NT Thyroid Liver Spleen Cardiac muscle NT Kidney Pancreas NT + Ovary NT Oviduct Testis + Bursa of Fabricius Results of 4 experiments. In the initial experiment all tissues were inoculated with the low dose of virus; in subsequent experiments the susceptible tissues were inoculated with the low dose, but the remainder were given the high dose. +, susceptible; i , doubtful susceptibility; -, not susceptible;
NT, not tested.
infection with 2-0 log1OCD50 of H120 strain is given in Table I. In a series of further experiments, the same range of tissues was inoculated with the H120 strain, but on these occasions 2 different doses were employed. Those tissues which were considered to be susceptible, as judged by the results of the first experiment, were inoculated with 2-0 log,OCD50 of virus as before. The tissues which had previously been designated as not susceptible were inoculated with a high dose of virus having a mean level over the series of 6-8 log,OCD50. As before, the fluids of the 10 tissue replicates of each type were pooled at 24-h intervals and assayed for the amounts of virus released each 24 h during the 96 h following inoculation. The results of 3
J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS
446
6-
4-
24
Nasal Turbinate
Trachea
0
Air Sac
1~~~~~~~
I
1
6-7 4i
cn
0
2-
Proventriculus
Lung o
I
-
l
Thyroid l
6-
44
2-
Kidney 0
24 48 72 96
Oviduct
Ovary 0
24
48
72
96
0
24
48
72
96
Hours FiG. 1. Daily release of virus into the maintenance medium (log10CD50/ml) of organ cultures of susceptible tissues inoculated with 2 -0 logjOCD50 of AIB virus (strain H120).
replicate experiments confirmed the original findings that 9 tissues were susceptible (see Table I) and the mean plots of their virus production are illustrated in Fig. 1. The upper respiratory tract tissues (nasal turbinates, trachea) responded the most rapidly, reaching maximum titres in the initial 24 h, whereas the lower tract tissues (air sac membranes, lung) attained comparable maximum virus production but within 48 to 72 h (see Fig. 1). Tissues originating from the urogenital tract were also
susceptible, including the kidney and oviduct which permitted virus replication comparable in timing and titre to the tissues of the lower respiratory system. The ovary produced virus only minimally in response to the low virus dose. The thyroid proved more efficient, however, and produced virus steadily throughout the period of study. A finding of note was that the mucosa of the proventriculus (or glandular stomach) could sustain virus replication to a significant degree in the same period.
INFECTION OF CHICKEN TISSUES WITH AIB VIRUS
447
j
Bursa
Conjunctiva , ,
-
[ I
--I~~~~~~~~~~~~
0 to
0
oa
0
6-
4-
2-
Oesophagus 0
24
48
72
96
24
48
78
96
Hours
FIG. 2.-Daily release of virus into the maintenance medium (log,OCD50/ml) of organ cultures of tissues inoculated with 6-2 log,OCD50 of AIB virus (strain H120). In an initial experiment, these types of tissue did not respond to inoculation with 2 0 log1OCD50 of the same virus.
When those tissues originally considered not to be susceptible were inoculated with a high dose of virus, the conjunctiva, caecal tonsil, testis and bursa of Fabricius each responded by yielding significant amounts of virus; the oesophagus and cloaca responded only minimally, and the remainder failed to produce virus at all (Table I). The results of assays of the virus production of these 6 tissues are illustrated in Fig. 2. In view of the aforegoing results further studies were made of the effect of 3 different virus in-puts in a limited range of susceptible tissues. Three groups of 10 replicates of each explant were infected with the H120 strain of AIB virus (0-2 ml) in the usual manner, the explants
of each group being inoculated with 7'0 log1oCD50, 44) log1OCD50 or 240 log10 CD50 respectively. The mean assay results for at least 2 replicate experiments of infecting 4 of the more susceptible tissues with 3 doses of virus are presented in Fig. 3, and those from similarly infecting 3 less susceptible tissues are given in Fig. 4. The lung, kidney and oviduct yielded comparable amounts of virus by 96 h irrespective of the level of in-put, whereas the proventriculus only responded when it received the high and medium doses. The testis, bursa of Fabricius and oesophagus supported virus replication following a high in-put in a manner analogous to the previous experiments
4483
J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS -
Kidney i 0
n
0
High
A-
cJ 0 C"
0
8
-
A
A 4
2
11
A
Oviduct 24
48
72
0
96
24
48
72
96
Hours FIG. 3.-Daily release of virus into the maintenance medium (log1OCD50/ml) of susceptible tissues inoculated with 3 in-put levels of AIB virus (strain H120).
(see Fig. 2), but in each case the medium dose produced a minimal response and the low dose produced no significant amounts of virus (Fig. 4). To test the possibility that differences in tissue susceptibility could vary according to the virulence of the strain of AIB virus, the HVI-9 strain was used to infect a series of tissue explants in a manner similar to the previous experiments. Based on the results obtained with the H120 strain, most tissues were inoculated
organ
cultures of 4
with either a low dose (2.0 log1OCD50) or a high dose (6.3 log1OCD50) of virus with the exception of 6 tissues which were inoculated with both the high and low doses. The results are given in Table II. The respiratory tract tissues and the oviduct were again susceptible to a low virus in-put, while the conjunctiva also responded to a low dose of this strain. The proventriculus mucosa, thyroid, kidney and ovary produced virus only in response to a high dose of the HVI-9
449
INFECTION OF CHICKEN TISSUES WITH AIB VIRUS
8Bursa
Oesophagus
6-
0
4-
von
Ca0 2
0
24
48
72
96
24
0
48
72
96
0
24
48
72
96
Hours FIG. 4. Daily release of virus into the maintenance medium (log,0CD50/ml) of organ cultures of 3 tissues, not originally designated as susceptible, inoculated with 3 in-put levels of AIB virus (strain H120).
strain. As with the H120 strain, the testis, bursa of Fabricius, caecal tonsil and cloaca were susceptible to a high dose. The tissues which were not susceptible to a high in-put of the H120 strain, with the
addition of the oesophagus, were not susceptible to a high dose of the HVI-9 strain.
Minimal infectious dose determinations The range of tissues selected for the TABLE II.-Susceptibility of Chicken Organ Cultures to High or Low Inocula of the determination of the MID50 was restricted to those susceptible to the H120 strain in HVI-9 strain of AIB virus the previous experiments. The MID50 Inoculum determinations were calculated from the 2-0 log10CD50 63 logCD50 Organ cultures results of assays of cultures 48 h after Conjunctiva + inoculation, the medium being changed Nasal turbinates NT + after the first 24 h. The period between Trachea NT + Air sac membranes NT + 24 and 48 h was considered to be the point Lung on the basis of monitoring virus when, NT Oesophagus Proventriculus mucosa decay in the controls, the presence of Gizzard mucosa NT unadsorbed residual virus could be disDuodenum NT counted. The mean results of at least 2 NT Large intestine _ Caecal tonsil NT replicate experiments are given in Table Caecum NT III. This list of tissues is arranged in ~+ Thyroid order of increasing MID50 requirements Liver NT NT Spleen for the initiation of a significant infection Cardiac muscle NT with the H120 strain of AIB virus. Kidney The tissues originating from the Pancreas Ovary respiratory system possessed in general the Oviduct NT + lowest MID50 requirements and that of the Testis NT + NT Bursa of Fabricius + nasal turbinates was comparable with the Cloaca trachea which in the present work was also Results of at least 2 experiments: +, susceptible; used as the standard assay system. The not susceptible; ±, doubtful susceptibility; air sac membranes had a somewhat NT, not tested. +
NT
-±
+
NT_
+
-,
450
J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS
TABLE III.-Minimal Infectious Dose although the visceral organs such as the (MID50) Requirement of Susceptible liver, pancreas and kidney showed discrete Chicken Tissues in Organ Culture using evidence of cloudy swelling and early the H120 strain of AIB virus degenerative necrosis in the centre of the explant. MID50 Tissue (logl0 CD50) In the infected cultures, over the same Nasal turbinates 0 3 the histological findings were little period, Trachea 0 3 different, with the principal exception of Air sac membranes 1*4 Lung 2 -2 the trachea. In the latter there was Oviduct 2 -2 progressive damage to the superficial Proventriculus mucosa 2-7 epithelial cells of the lumen accompanied Conjunctiva Kidney 3 9 by exfoliation of the ciliated cell layer, an 4-6 Ovary accumulation of cell debris in the lumen Bursa of Fabricius and a general thinning of the epithelial Thyroid 4*8 Testis 4.9 lining. A very limited cellular migration Caecal tonsil 5.9 and a number of pyknotic nuclei were both 5-9 Cloaca Oesophagus 6-1 apparent, although the basal cell layer Mean results of at least 2 replicate experiments. remained intact. The deeper layers of the Mean virus in-put 6 6 logl0 CD 50/0 * 2 ml. tracheal ring, including the cartilaginous tissue, remained unaltered. the intestinal tissues, the mucosa higher MID50 value, but this in turn was wasInundamaged and its structure was well lower than that of the lung, which had the preserved. The epithelial linings were highest value of any respiratory tissue. intact throughout and no lesions were The oviduct possessed a comparable evident in the mucosa of the provenMID50 value to the lung, and the proven- triculus. triculus mucosa could be infected successIn the case of the other infected fully with an average of only 0 5 log10 CD50 organs, the liver showed incipient necrosis more virus. The conjunctiva can be said of the central area, extending to about to be associated with the respiratory tract one-third of the explant within 48 h and by way of the nasal cavity, but, in contrast to approximately two-thirds by 96 h, to the true respiratory tract tissues, its with a rim of apparently healthy tissue MID50 requirement was distinctly higher surrounding it. A similar process, but to than any of them, and comparable with a lesser extent, was noted in the kidney, that of the kidney. A group of tissues pancreas, spleen and lung. In each case, comprising the ovary, bursa of Fabricius, no specific pathological changes could be thyroid and testis all had a similar facility confidently ascribed to the results of AIB for infection by the H120 strain, whereas virus infection. Furthermore, no evithe caecal tonsil, cloaca and oesophagus dence was observed of any cellular had significantly increased MID50 figures, dedifferentiation in any of the tissue the latter possessing the highest for all the explants. tissues under investigation. 3-7
4.7
DISCUSSION
Histological examinations In the haematoxylin and eosin-stained sections of the explants which were left uninoculated for a period of 96 h, the histological architecture was generally not visibly affected and was apparently well maintained. Hollow organs such as the trachea and intestine showed no change,
The technique of using roller tubes for the culture of chicken tracheal explants is an established practice (Harnett and Hooper, 1968; Cherry and TaylorRobinson, 1970; Butler et al., 1972; Darbyshire et al., 1975; Cook et al., 1976) and in the present studies was extended to the maintenance of explants from other
INFECTION OF CHICKEN TISSUES WITH AIB VIRUS
tissues, as it was considered preferable to the conventional use of Petri dishes as described by Hoorn (1964). The explants were bathed regularly by the maintenance fluid during rolling which resulted in the retention of an adequate cellular architecture within infected or uninfected tissues and in satisfactory virus production. The apparent absence of histological changes directly attributable to AIB virus infection implies that specific viral damage of cells was minimal. It is possible that, despite their paucity, any specific cytopathological changes would be obscured by the natural degenerative processes occurring in the explants as culture continued. The H120 strain is widely employed as a live vaccine in disease control, and provides a convenient model for the study of the pathogenesis of AIB virus. The results obtained here from a survey of the susceptibility of a range of chicken organs indicate that tissues of the respiratory system, particularly those of the upper part of the tract, are more susceptible to AIB virus infection than those from other sites. This is not unexpected in view of the findings of a number of workers who have studied the experimental disease in chickens. Kawakubo et al. (1961) infected chickens with the CAV strain of AIB virus by various routes and recovered it in highest titre in the tracheal mucus and from the lungs. Virus was also found in intestinal contents for up to 21 days after infection but active infection of any specific site was not described. Hofstad and Yoder (1966) administered a number of strains to chickens by aerosol and subsequently recovered them in greatest amounts from the trachea, lung and air sacs, although virus was also present in lower titres in the kidney, pancreas, spleen, liver and the bursa of Fabricius. Doherty (1967b) assayed virus from tissues of infected chickens and recovered it for up to 7 days, mostly from the lungs and trachea, as well as from the kidney, spleen, bursa of Fabricius, proventriculus, liver and heart.
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In each of the aforegoing investigations (Kawakubo et al., 1961; Hofstad and Yoder, 1966; Doherty, 1967b) a viraemia was demonstrated, although there is a lack of agreement as to its timing or duration. The collection of tissues which may contain infective blood could lead to an apparent demonstration of virus in otherwise insusceptible tissues. In this respect the findings with the infection of explants in vitro may reflect more accurately the early events in the pathogenesis of AIB virus infection in chickens. In interpreting the results from the present work it is assumed that a lack of susceptibility of tissues in culture reflects their insusceptibility in vivo. In a brief report, Jones (1973) has stated that when the M41 (Massachusetts) strain of AIB virus was used to infect a limited number of chicken organ cultures, it replicated well in the trachea and oviduct, but only at a low level in lung, kidney, spleen and small intestine. When another chicken was used only the trachea responded to infection and this was ascribed to individual variation. In the present studies, an attempt has been made to obviate such variation by replicate sampling involving numbers of chickens of a similar age. The MID50 values provide a quantitative and relative assessment of the efficiency with which AIB virus would initiate the infection of various tissues in the susceptible chicken. These values correlate with the results of the surveys for tissue susceptibility and confirm that the upper part of the respiratory tract was the most vulnerable and more so than the air sac membranes, followed in turn by the lungs and oviduct which were themselves both comparable. The oesophagus was the least easily infected (see Fig. 2 and 4). In most respects the results of infecting tissues with the HVI-9 strain were similar to those obtained with the H120 strain, when used at 2 levels of in-put, with the exception of the thyroid, kidney and ovary, due possibly to differences in strain pathogenicity (Table II). The isolation
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J. H. DARBYSHIRE, J. K. A. COOK AND R. W. PETERS
of AIB virus from the kidneys of chickens infected experimentally has been reported frequently (Kawakubo et al., 1961; Hofstad and Yoder, 1966; Doherty 1967b; Cumming 1969), although certain strains have a definitive tropism for that organ in field cases (Cumming, 1962, 1963, 1969). The significance of strain as well as serotype differences in artificial infections of organ cultures is accordingly now under further investigation. The susceptibility of the oviduct to both strains (H120, HVI-9), together with its comparability in virus production with tissues from the lower respiratory tract, reflects its involvement in the natural disease. Experimental infection of dayold chicks or laying hens can lead to abnormalities of the reproductive tract and variations in egg quality (Crinion, Ball and Hofstad, 1971a; Jones and Jordan, 1972; McDougall, 1968), for the virus infects the epithelial cells of the oviduct of young chicks or older birds, probably as the result of a viraemia, and may be recovered from that site for up to 11 days after experimental infection (Crinion et al., 1971b; Jones and Jordan, 1971). The susceptibility of the proventriculus mucosa accords with the recovery of virus from that site in chickens infected experimentally by Doherty (1967b) who considered that it may have been associated with virus in ingested tracheal mucus. The ability of the mucosa to be infected in culture suggests that it could acquire infection in vivo either by ingestion or as the result of a viraemia. The definitive reaction of the cloaca to a high in-put of the HVI-9 strain and the lesser one to the H120 strain implies that each could initiate infection of that organ. This supports the findings of Hudson and Beaudette (1933) that birds can be infected experimentally via the cloaca, so that this organ must be regarded as a potential portal of infection in vivo. The more susceptible tissues, such as lung, oviduct and kidney, each produced comparable virus yields by 96 h when
different inocula were used (see Fig. 3), and there may be a similar course of events in vivo with such tissues whatever the level of infection. This was also the case, in part, with the proventriculus mucosa, but its poor response in this series of experiments to the low dose of virus in view of the previous findings (see Fig. 1) needs further investigation. The responses of the less susceptible tissues, like testis, bursa of Fabricius and oesophagus, indicate that, with the H120 strain at least, a threshold level of virus is required before these tissues will respond to infection (Fig. 4). The establishment of the infection of organs with AIB virus in natural infections in vivo depends on a number of factors, including numbers and availability of susceptible cells as well as differences in pathogenicity between virus strains. The respiratory tract is the obvious target organ, the present results showing that it is the easiest to infect and, in the upper part, produces virus the most rapidly. This provides epidemiological supportive evidence for the rapid spread of virus within a population of susceptible birds. In this respect, a primary infection of the conjunctiva per se would appear to play a less significant role. If the respiratory tract were not infected initially, then alternatively various parts of the alimentary tract could permit virus production, including the proventriculus mucosa. The innate resistance of other parts of the alimentary tract must be due to an unavailability of susceptible cells, in view of the insusceptibility of washed explants, rather than to adverse chemical and pH conditions or the presence of deleterious enzymes. The importance of local production of AIB virus in the alimentary tract is exemplified by the finding by Pette (1959) that virus could be recovered from the cloacal contents for up to 24 days following oral infection. The infection of certain organs in vivo would rely on the appearance of circulating virus. In low titres the latter would presumably infect those tissues considered susceptible
INFECTION OF CHICKEN TISSUES WITH AIB VIRUS
to a low virus in-put in this work, whereas circulating virus in high titre would further infect such tissues as the testis, bursa of Fabricius and oesophagus. Other factors in the pathogenesis of AIB virus infection in vivo concern the availability of cells for virus adsorption and penetration, and the circumvention of such local barriers as antibody and cellular mechanisms. Organ cultures provide models of less complexity than do experimentally infected chickens, since local antibody production and cellular defence activities are probably minimal or in abeyance. The infection of organ cultures differs, moreover, from that of tissue culture monolayers in so far as the cells in an explant are fully differentiated and would possess a range of varying susceptibility. The role of interferon and the effects of its possible differential production by the various cell types are also aspects for further consideration. In the present investigations the use of organ cultures to examine and quantify the efficiency of AIB virus infection of chicken tissues has now been established. REFERENCES BEACH, J. R. & SCHALM, 0. W. (1936) A Filterable Virus Distinct from that of Laryngotracheitis, the Cause of a Respiratory Disease of Chicks. Poultry Sci., 15, 199. BROADFOOT, D. I. & SMITH, W. M. (1954) Effects of Infectious Bronchitis in Laying Hens on Egg Production, per cent Unsettable Eggs and Hatchability. Poultry Sci., 33, 653. BUTLER, M., ELLAWAY, W. J. & HAALL, T. (1972) Comparative Studies on the Infectivity of Avian Respiratory Viruses for Eggs, Cell Cultures and Tracheal Explants. J. comp. Path., 82, 327. CHERRY, J. D. & TAYLOR-RoBINsoN, D. (1970) Large Quantity Production of Chicken Embryo Tracheal Organ Cultures and Use in Virus and Mycoplasma Studies. Appl. Microbiol., 19, 658. COLWELL, W. M. & LUKERT, P. D. (1969) Effects of Avian Infectious Bronchitis Virus (IBV) on Tracheal Organ Cultures. Avian Di8., 13, 888. COOK, J. K. A. (1968) Duration of Experimental Infectious Bronchitis in Chickens. Re8. vet. Sci., 9, 506. COOK, J. K. A., DARBYSHIRE, J. H. & PETERS, R. W. (1976) The Use of Chicken Tracheal Organ Cultures for the Isolation and Assay of Avian Infectious Bronchitis Virus. Archiv. Virol., 50, 109. CRINION, R. A. P., BALL, R. A. & HOFSTAD, M. S. (1971a) Pathogenesis of Oviduct Lesions in Immature Chickens Following Exposure to
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Infectious Bronchitis Virus at One Day Old. Avian Di8., 15, 32. CRINION, R. A. P., BALL, R. A. & HOFSTAD, M. S. (1971b) Abnormalities in Laying Chickens Following Exposure to Infectious Bronchitis Virus at One Day Old. Avian Dis., 15, 42. CUMMING, R. B. (1962) The Aetiology of Viraemia of Chickens. Au8t. vet J., 38, 554. CUMMING, R. B. (1963) Infectious Avian Nephrosis (Uraemia) in Australia. Au8t. vet. J., 39, 145. CUMMING, R. B. (1969) Studies on Avian Infectious Bronchitis Virus. I. Distribution and Survival of the Virus in Tissues of Affected Chickens and Studies on the Carrier Status. Au8t. vet. J., 45, 305. DARBYSHIRE, J. H., COOK, J. K. A. & PETERS, R. W. (1975) Comparative Growth Kinetic Studies on Avian Infectious Bronchitis Virus in Different Systems. J. comp. Path., 85, 623. DELAPLANE, J. P. & STEWART, H. 0. (1939) Studies of Infectious Bronchitis. Bull. Rhode Ila. agric. Exp. Stn., 237. DOHERTY, P. C. (1967a) Occurrence of Avian Infectious Bronchitis Virus in the Tissues of Experimentally Infected Chickens. Qd. J. Agric. anim. Sci., 24, 75. DOHERTY, P. C. (1967b) Titration of Avian Infectious Bronchitis Virus in the Tissues of Experimentally Infected Chickens. Au8t. vet. J., 43, 575. HARNETT, G. B. & HOOPER, W. L. (1968) Test-tube Organ Cultures of Ciliated Epithelium for the Isolation of Respiratory Viruses. Lancet, i, 339. HIGGINS, P. G. (1966) The Isolation of Viruses from Acute Respiratory Infections. V. The Use of Organ Cultures of Human Embryonic Nasal and Tracheal Epithelium. Mon. Bull. Mini8t. Hlth., 25, 283. HOFSTAD, M. S. & YODER, H. W. (1966) Avian Infectious Bronchitis-Virus Distribution in Tissues of Chicks. Avian Di8., 10, 230. HOORN, B. (1964) Respiratory Viruses in Model Experiments. Transaction of the 15th Congress of the Scandinavian Oto-laryngological Society 1963. Acta oto-lar., 188, Suppl., 138. HOORN, B. (1966) Organ Cultures of Ciliated Epithelium for the Study of Respiratory Viruses. Acta path. microbiol. 8cand., 183, Suppl. 66. HOORN, B. & TYRRELL, D. A. J. (1969) Organ Cultures in Virology. Prog. med. Virol., 11, 408. HUDSON, C. B. & BEAUDETTE, F. R. (1933) The Susceptibility of Cloacal Tissue to the Virus of Infectious Bronchitis. Cornell Vet., 23, 63. JOHNSON, R. B. & NEWMAN, J. A. (1971) Measurement of Antigenic and Pathogenic Variability of Infectious Bronchitis Virus Strains by Trachea Culture. Avian Di8., 15, 233. JOHNSON, R. B., NEWMAN, J. A. & WILLS, F. K. (1969) Titration of Infectious Bronchitis Virus in Tracheas of Susceptible and Immune Chickens. Avian Di8., 13, 632. JONES, R. C. (1973) The Use of a Combined Immunofluorescence-Organ Culture System for Studying Infectious Bronchitis Virus. Proc. Wld Vet. Poult. As8oc., 2, 780. JONES, R. C. & JORDAN, F. T. W. (1971) The Site of Replication of Infectious Bronchitis Virus in the Oviduct of Experimentally Infected Hens. Vet. Rec., 89, 317. JONES, R. C. & JORDAN, F. T. W. (1972) Persistence of Virus in the Tissues and Development of the
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Oviduct in the Fowl Following Infection at Day Old with Infectious Bronchitis Virus. Re8. vet. Sci., 13,52. KAWAKUBO, A., MIYAMOTO, T. & NAKAMURA, J. (1961) Studies on Infectious Bronchitis of Chickens. III. Experiments with Artifically Infected Chickens. N.I.B.S. Bull. biol. Re8., 6, 1. MCDOUGALL, J. S. (1968) Infectious Bronchitis in Laying Fowls. Its Effect upon Egg Production and Subsequent Egg Quality. Vet. Rec., 83, 84. PETTE, J. (1959) Zur Ausscheidung des Huhnerbronchitisvirus im Kloakeninhalt. Mh. Tierheilk., 11, 296. REED, L. J. & MUENCH, H. (1938) A Simple Method of Estimating Fifty per Cent End Points. Am. J. Hyg., 27, 493. SEVOIAN, M. & LEVINE, P. P. (1957) Effects of Infectious Bronchitis on the Reproductive Tracts,
Egg Production and Egg Quality of Laying Chickens. Avian Di8., 1, 136. SILLER, W. G. & CUMMING, R. B. (1974) The Histopathology of an Interstitial Nephritis in the Fowl Produced Experimentally with Infectious Bronchitis Virus. J. Path., 114, 163. ToMs, G. L., RoSZTOCZY, I. & SMITH, H. (1974) The Localization of Influenza Virus: Minimal Infectious Dose Determinations and Single Cycle Kinetic Studies on Organ Cultures of Respiratory and Other Ferret Tissues. Br. J. exp. Path., 55, 116. TYRRELL, D. A. J. & BYNOE, M. L. (1965) Cultivation of a Novel Type of Common-Cold Virus in Organ Cultures. Br. med. J., i, 1467. TYRRELL, D. A. J. & BYNOE, M. L. (1966) Cultivation of Viruses from a High Proportion of Patients with Colds. Lancet, i, 76.
