Pathogenesis of conjunctivitis caused by Newcastle disease viruses in specific-pathogen-free chickens

Abstract

The pathogenesis of conjunctivitis caused by Newcastle disease viruses (NDVs) was investigated in 3-week-old specific-pathogen-free chickens. The chickens were inoculated intramuscularly, by eye drop or intranasally, with 10⁷ plaque forming units of viscerotropic velogenic Newcastle disease virus (VVNDV), mesogenic NDV or lentogenic NDV. Macroscopically, lower palpebral conjunctivae appeared red with oedematous swelling in chickens inoculated with VVNDV. Histologically, mild lesions were focal hyperplasia of the conjunctival epithelial cells with cellular infiltration in the lamina propria of the conjunctivae in the chickens inoculated with VVNDV. Moderate lesions were vascular necrosis with congestion and haemorrhages, hyperplasia of conjunctival epithelial cells, and oedema and cellular infiltration in the lamina propria of the conjunctivae. Fibrin thrombi were observed in the capillaries of the lamina propria. In marked lesions, the whole conjunctiva was affected with more severe and extensive lesions. The mesogenic and lentogenic NDV strains induced no gross conjunctival lesions, but induced mild conjunctivitis without vascular necrosis.

Introduction

Newcastle disease, a serious world-wide poultry disease, is caused by Newcastle disease virus (NDV), a member of the genus Rubulavirus, family Paramyxoviridae. NDV is divided into five pathotypes (Alexander, 2003). Viscerotropic velogenic Newcastle disease (Doyle's form), is a very acute and lethal form of Newcastle disease with haemorrhagic lesions of the digestive tract. Neurotropic velogenic Newcastle disease (Beach's form) had neurological and respiratory lesions. Mesogenic Newcastle disease (Beaudette's form) is an acute respiratory and sometimes lethal nervous infection of young chicks. Mortality is rare in older birds. Lentogenic Newcastle disease (Hitchner's form) is a mild or inapparent respiratory infection of chickens. The asymptomatic-enteric form (Ulster type) manifests chiefly as gut infections with lentogenic viruses, causing no obvious disease.

Lymphoid, vascular, respiratory, neural, and reproductive lesions are seen in chickens as pathological features of Newcastle disease (Alexander, 1997). It is also well known that NDV causes conjunctivitis (Cheville et al., 1972; Spalatin et al., 1973; Katoh, 1977; Alexander, 2003) and induces conjunctivitis in human (Hales & Ostler, 1973). In the latter report, an epidemic of Newcastle disease occurred in turkeys in 1965 and 1966 in the United States, and several workers in close contact with the turkeys at the processing plant developed a follicular conjunctivitis and a rise of antibodies against NDV. Therefore, Newcastle disease is one of a few chicken zoonotic diseases. Clinically, conjunctivitis is a significant sign of Newcastle disease in chickens. There are no histopathological studies on NDV-induced conjunctivitis.

This paper investigates the pathogenesis of conjunctivitis in 3-week-old specific-pathogen-free (SPF) chickens inoculated with NDV strains of different virulence.

Materials and Methods

Chickens

The 3-week-old chickens used for this study were obtained from an SPF White Leghorn flock (line 15I) maintained at our laboratory (Furuta et al., 1980). The flock was negative for NDV and 10 other avian viruses (Furuta et al., 1980). The chickens were kept in the isolators under negative pressure and supplied with filtered air throughout the experimental period.

Newcastle disease virus

Five strains of NDV were used in this study (Table 1). The JP/Chiba/85, JP/Ibaraki/85 and JP/Okinawa/91 strains (Viscerotropic velogenic Newcastle disease viruses [VVNDVs]) were isolated from chickens with Newcastle disease in Japan (Imai et al., 1986). The JP/TY-1/85 strain, a mesogenic NDV, was isolated from a chicken with non-purulent encephalomyelitis in Japan (Shirai et al., 1986). The Mukteswar strain (a mesogenic vaccine strain) and the B1 strain (a lentogenic vaccine strain) were used (McMillan et al., 1986). According to typing of viral genes, JP/Chiba/85 (Mase et al., 2002) belongs to type VII, JP/Ibaraki/85 (Mase et al., 2002) to type VI, JP/Okinawa/91 (Mase et al., 2002) to type VIII, JP/TY-1/85 (Mase et al., 2002) to type VI, Mukteswar (GenBank accession number AY117021) to type IV, and B1 (GenBank accession number U22266) to type II.

Table 1. Morbidity rates and lesion scores of conjunctivitis in chickens inoculated with NDV

							Histological conjunctivitis
							Mean lesion scores
							Lower palpebral conjunctiva		Upper palpebral conjunctiva
Experiment	Group^a	NDV strain	Virulence^b	Genotype	Inoculation route^c	Morbidity rate (%)	Right	Left	Right	Left
1	1	JP/Chiba/85	VV	VII	IM	100	1.1 (V, 0.7)^e	1.0 (V, 0.8)	0.6 (V, 0.5)	0.3 (V, 0.4)
	2	JP/Chiba/85	VV	VII	IN	100	1.7 (V, 2.3)	1.2 (V, 1.3)	0.5 (V, 0.8)	0.2 (V, 0.3)
	3	JP/Chiba/85	VV	VII	ED	100	2.4 (V, 2.6)	1.7 (V, 2.0)	1.0 (V, 1.2)	0.7 (V, 0.7)
	4	JP/TY-1/85	M	VI	IN	10	0.3 (V, 0.1)	0.0 (V, 0)	0.0 (V, 0)	0.0 (V, 0)
	5	JP/TY-1/85	M	VI	ED	0	0.1 (V, 0)	0.0 (V, 0)	0.0 (V, 0)	0.0 (V, 0)
2	6	JP/Ibaraki/85	VV	VI	ED	100	3.0(V, 2.8)	1.1(V, 0)	2.0 (V, 1.7)	0.7 (V, 0.1)
	7	JP/Okinawa/91	VV	VIII	ED	100	2.5 (V, 2.1)	1.5 (V, 0)	1.4 (V, 1.6)	0.5 (V, 0)
	8	Mukteswar	M	IV	ED	100	2.0 (V, 0)	0.2 (V, 0)	1.1 (V, 0)	0.1 (V, 0)
	9	B1	L	II	ED	100	1.2 (V, 0)	0.1 (V, 0)	0.8 (V, 0)	0.0 (V, 0)
	10	Control	–	–	–	0	0.0 (V, 0)	0.0 (V, 0)	0.0 (V, 0)	0.0(V, 0)
^a Number of birds each group was 10.
^b VV, viscerotropic velogenic; M, mesogenic; L, lentogenic.
^c IM, intramuscularly; IN, intranasally; ED, via eye drop.
^d Lesion scores: 0=no lesons, 1=mild, 2=moderate, 3=severe lesions.
^e Mean lesion score of conjunctivitis (mean lesions score of vascular necrosis); V, vascular necrosis.

These viruses were cultured in chicken kidney cells. Cell cultures were prepared according to the method described by Kawamura (1961).

Experimental design

In Experiment 1, the chickens were divided into five groups of 10 birds (Table 1); group 1, JP/Chiba/85 strain, intramuscularly; group 2, JP/Chiba/85, intranasally; group 3, JP/Chiba/85, via eye drop into the right eye; group 4, JP/TY-1/85, intranasally; and group 5, JP/TY-1/85, via eye drop into the right eye. The birds in each group were inoculated with 0.1 ml inoculum containing 10⁷ plaque-forming units NDV. Chickens that died were necropsied within 3 h of death.

In Experiment 2, chickens were divided into five groups with 10 birds each group (Table 1); group 6, JP/Ibaraki/85; group 7, JP/Okinawa/91; group 8, Mukteswar; group 9, B₁; and group 10, uninoculated control. The chickens of groups 6 to 9 in Experiment 2 were inoculated via eye drop with 0.1 ml inoculum containing 10⁷ plaque-forming units NDV into the right eye. The chickens were humanely killed using CO₂ and necropsised at 3 days after inoculation. Chickens that died during the observation period were necropsied within 3 h of death.

Histopathology

Following postmortem examination of the chickens, the liver, heart, spleen, kidneys, lung, trachea, bursa of Fabricius, thymus, oesophagus, gastrointestinal tract, pancreas, femur, peripheral nerve, and brain were removed and were fixed in 10% buffered formalin. After removing the brain, the heads were fixed in 10% buffered formalin and then decalcified, and transverse sections containing the turbinates, infraorbital sinus, eyeballs including conjunctivae, and air spaces of the posterior skull were cut. All tissue samples were then embedded in paraffin, sectioned at 4 μm, and stained with haematoxylin and eosin.

Mean lesion scores of histological lesions

The lesion scores of conjunctivitis and vascular lesions were assigned according to the following criteria; 0=no lesions; 1=mild lesions; 2=moderate lesions; 3=severe lesions. Mean lesion scores were calculated by adding the lesion scores and dividing the number of chickens observed for microscopic evaluation.

Results

Clinical signs

In Experiment 1, all the chickens of groups 1 to 3 (inoculated intramuscularly, intranasally, or via eye drop, respectively, with JP/Chiba/85) exhibited depression and sometimes respiratory signs, and died at 3 days after inoculation (mortality rates of groups 1 to 3 were 100%). In group 4 (inoculated intranasally with JP/TY-1/85), one bird was depressed and died at 3 days after inoculation (the mortality rate was 10%). The chickens of group 5 (inoculated via eye drop with JP/TY-1/85) had neither clinical signs nor mortality. In Experiment 2, groups 6 and 7 (inoculated via eye drop with JP/Ibaraki/85 and JP/Okinawa/91, respectively) had no mortality but all chickens were moribund at 3 days after inoculation. Groups 8 and 9 (inoculated via eye drop with Mukteswar and B1, respectively) and group 10 (uninoculated control) had no mortality and no birds exhibited clinical signs.

Gross pathology

There were few lesions in the bulbar conjunctiva in any chickens, so the conjunctival lesions described in the following were found in the palpebral conjunctiva. Gross conjunctival lesions were observed in the chickens of groups 1 to 3, 6 and 7 inoculated with VVNDV. There was reddening and swelling of the lower conjunctiva (Figure 1). The conjunctival lesions were usually seen bilaterally. The lesions on the inoculated side were more severe than on the opposite side. There were multifocal white foci on the spleen, and occasionally haemorrhages in the mucosa of the proventriculus, duodenum, and caecal tonsil in the affected chickens inoculated with VVNDV. Atrophy of the lymphoid organs (bursa of Fabricius and thymus) was observed particularly in dead chickens inoculated with VVNDV.

Figure 1. Conjunctival congestion and haemorrhages (arrows) in a chicken inoculated intramuscularly with the JP/Chiba/85 of VVNDV. The chicken died 3 days after inoculation.

Histopathology

The incidence of lesions scores of conjunctivitis in the chickens inoculated with NDV are summarized in Table 1. The more severe of conjunctival lesions in the palpebra were observed in the chickens inoculated with VVNDV when compared with those inoculated with mesogenic NDV and lentogenic NDV, although morbidity rates of histological conjunctivitis were 100% in the groups inoculated with VVNDV, mesogenic NDV, and lentogenic NDV. However, the JP/TY-1/85 strain, neurotropic mesogenic NDV, was rarely pathogenic for the conjunctiva. The lesions of the lower palpebra were more severe than those of the upper palpebra.

Control birds had occasional lymphoid aggregates, in the palpebral conjunctiva, but none of the other changes described under lesion scores.

Mild to severe lesions were seen in the conjunctiva of chickens inoculated with VVNDV. In mild lesions, mild lymphocytic infiltration, oedema, sloughing of epithelial cells and vascular congestion were observed in the base of the conjunctiva (Figure 2). In moderate lesions, vascular necrosis, congestion and haemorrhages, fibrin thrombi, oedema, heterophilic infiltration and proliferation of macrophages were observed. Such lesions were also frequently seen in the third palpebra (nictitating membrane). There were hyperplasia and desquamation of the conjunctival epithelial cells (Figures 3 and 4), vascular congestion and haemorrhages, vascular fibrinous necrosis (Figure 3), fibrin thrombi (Figure 4), oedema (Figures 3 and 4), heterophilic infiltration, and proliferation of macrophages containing debris (Figures 3 and 4) in the lamina propria of conjunctiva. In severe lesions, more extensive areas were affected by changes similar to those cited earlier (Figure 5). In the infraorbital sinus (Figure 6) and the nasal turbinate (Figure 7), fibrinous necrosis of the blood vessels, oedema, heterophilic infiltration and proliferation of macrophages in the lamina propria were observed. There was depletion of plasma cells in the lamina propria and hydropic degeneration of the surface epithelium in Harderian glands. There were no lesions in any parts of the eyeballs of the chickens inoculated with VVNDV.

Figure 2. Mild lesions in the conjunctiva (arrows) of a chicken inoculated intramuscularly with JP/Chiba/85 of VVNDV. The chicken died 3 days after inoculation. Cellular infiltration and hyperplasia of the conjunctival epithelial cells in the base area of conjunctiva. The palpebra (P), third palpebra (nictitating membrane) (N), and scleral ossicle (S) are seen. Haematoxylin and eosin. Bar=800 μm.

Figure 3. Fibrinous vascular necrosis (arrows) in moderate lesions of the conjunctiva of a chicken inoculated via eye drop with JP/Chiba/85 of VVNDV. There are also hyperplasia and desquamation of conjunctival epithelial cells, oedema, and heterophilic infiltration in the lamina propria. The chicken died 3 days after inoculation. Haematoxylin and eosin. Bar=200 μm.

Figure 4. Fibrin thrombi (arrows) in the blood capillaries in moderate lesions of conjunctiva of a chicken inoculated via eye drop with JP/Chiba/85 of VVNDV. Desquamation of conjunctival epithelial cells and heterophilic infiltration in the lamina propria are seen. The chicken died 3 days after inoculation. Haematoxylin and eosin. Bar=200 μm.

Figure 5. Severe lesions in the conjunctiva of a chicken inoculated intranasally with JP/Chiba/85 of VVNDV. The chicken died 3 days after inoculation. Marked congestion, haemorrhage, cellular infiltration, oedema and vascular necrosis are seen diffusely throughout the palpebra. The palpebra shows marked swelling. Haematoxylin and eosin. Bar=800 μm.

Figure 6. Infraorbital sinus of a chicken inoculated intranasally with JP/Chiba/85 of VVNDV. Diffuse infiltration of heterophils, vascular necrosis (arrow), and oedema are seen in the lamina propria. Exudats includes fibrin and heterophils, is present in the sinus lumen. The chicken died 3 days after inoculation. Haematoxylin and eosin. Bar=200 μm.

Figure 7. Nasal turbinate of a chicken inoculated intranasally with JP/Chiba/85 of VVNDV. The chicken died 3 days after inoculation. There is vascular necrosis (arrow), oedema, and heterophilic infiltration in the lamina propria. Haematoxylin and eosin. Bar=100 μm.

No lesions or mild lesions were seen in the conjunctiva of chickens inoculated with lentogenic or mesogenic NDV strains.

In other tissues, there were characteristic lesions in the VVNDV infections; splenic necrosis, and lymphoid depletion in lymphoid organs with congestion, haemorrhages, and heterophilic infiltration. There were no lesions other than conjunctivitis in chickens inoculated with the mesogenic or lentogenic NDV, except for one dead bird inoculated with JP/TY-1/85 that exhibited splenic necrosis.

Discussion

VVNDV-induced conjunctival changes included the fibrinous necrosis of the blood vessels, fibrin thrombi, congestion, haemorrhages, oedema, heterophilic infiltration, proliferation of macrophages, and conjunctival epithelial cell hyperplasia and desquamation. Of these changes, damage to blood vessels may be important for the development of moderate to severe conjunctivitis of chickens inoculated with viscerotropic velogenic Newcastle disease.

The VVNDV stains used in this study could induce macroscopically evident conjunctivitis. The strains belonging to mesogenic and lentogenic NDV could induce no macroscopic changes but there was mild histological conjunctivitis. These differences may be due to pathogenic effects of NDV on the blood vessels in the conjunctiva. We speculate that the mesogenic and lentogenic NDV have little pathogenicity for the blood vessels in the conjunctiva, although studies on the pathogenic effects of more strains of mesogenic and lentogenic NDV on the conjunctiva in the chicken are necessary. We also suggest that the pathogenic mechanism of conjunctivitis induced by VVNDV in SPF chickens may be vascular necrosis in the lamina propria of conjunctivae.

Conjunctivitis was seen in the chickens inoculated by various routes with NDV. This suggests that NDV has a high affinity for the conjunctiva. In addition, it is suggested that the conjunctiva of the lower palpebra was more susceptible than that of the upper palpebra for NDV. This may be because the blood vessels in the lower palpebra are distributed more extensively than in the upper palpebra.

It is interesting that the JP/TY-1/85 strain could induce few histological changes in the conjunctiva, even when the chickens were inoculated via eye drop. This strain is neurotropic (Shirai et al., 1986) and may have no affinity for the conjunctiva.

Virulent avian influenza virus infection and VVNDV infection, acute fatal diseases with systemic haemorrhages in the chickens, are very important diseases of the poultry industry. It is necessary to diagnose and differentiate them as rapidly and correctly as possible. There are few reports on conjunctivitis induced by virulent avian influenza (Uys & Becker, 1967), while there are many reports on macroscopical NDV-associated conjunctivitis (Kawasaki et al., 1968; Cheville et al., 1972; Spalatin et al., 1973; Katoh, 1977; Alexander, 2003; Brown et al., 1999). Detection of conjunctivitis with vascular necrosis may thus be useful in the diagnosis of viscerotropic velogenic Newcastle disease infection in the chickens, although further more pathological studies of chickens infected with NDV are necessary.

Acknowledgements

The authors thank Dr Y. Ando and Mr T. Fujisawa for providing photographs, Mr M. Kobayashi and Miss Megumi Shimada for histological and immunohistochemical assistance, and Miss Naomi Oka for helpful assistance in preparing the manuscript.