**2.1 Viruses and cells**

588 A Bird's-Eye View of Veterinary Medicine

2006). NDV strains are classified into low virulent (lentogenic), moderately virulent (mesogenic), and highly virulent (velogenic) pathotypes based on severity of disease produced in chickens. In contrast very little is known about pathogenicity of APMV-2 to -9 in chickens and turkeys. APMV-2 has been shown to cause mild disease and drop in egg production in chickens and turkeys (Warke et al., 2008). APMV-6 and -7 have been associated with respiratory disease in turkeys (Shortridge et al., 1980; Saif et al., 1997). APMV-4, -8 and -9 have been isolated from different species of birds but the clinical signs of the disease in those birds were not apparent (Alexander et al., 1983; Gough and Alexander, 1984; Stallknecht et al., 1991; Maldonado et al., 1995; Capua et al., 2004). Recently, experimental infection of 1-day-old chicks with APMV-2, -4 and -6 showed viral infection in

APMV-3 has been isolated from wild and domesticated birds in different parts of the world (Tumova et al., 1979b; Alexander, 1980b; Macpherson et al., 1983; Alexander, 2003). Recently, APMV-3 was isolated from ostrich, indicating a wide host range for the virus (Kaleta et al.). The virus has been isolated from diseased turkeys associated with coughing, nasal discharge and swelling of the infra-orbital sinus (Redmann et al., 1991). APMV-3 has been associated with encephalitis and high mortality in caged birds (Tumova et al., 1979a). The virus causes acute pancreatitis and central nervous system (CNS) symptoms in *Psittacine* and *Passerine* birds (Beck et al., 2003). APMV-3 also infects chickens at an early age, with evidence of stunting growth that may be more marked in broiler chicken breeds (Alexander and Collins, 1982a). In terms of pathogenicity to domestic poultry birds, APMV-3 probably is second in importance to NDV. The exact economic impact of APMV-3 infection in poultry industry is not known. This is partly because the pathogenicity of APMV-3 in poultry is not well studied. There is a high degree of amino acid sequence variation between APMV-3 and APMV-1 (NDV), but by the HI test there is low level of cross reaction between APMV-3 and APMV-1 serum samples, which often leads to

APMV-3 strain Wisconsin was first isolated from a turkey in 1968 in Wisconsin (Tumova et al., 1979b). APMV-3 strain Netherlands was isolated from a parakeet in 1975 in the Netherlands and is the prototype for the entire serotype (Alexander and Chettle, 1978). Initially, these two viruses were considered as two different strains of APMV-3 based on cross HI test using monoclonal antibodies (Anderson et al., 1987; Anderson and Russell, 1988). Recently, reciprocal cross HI and cross neutralization assays using post infection serum from chicken indicated that these two strains are antigenically distinct, although the difference was modest (Kumar et al., 2010b). However, complete genome sequence analysis revealed substantial genome-wide nucleotide and amino acid sequence differences that are consistent with the two strains representing distinct antigenic subgroups (Kumar et al., 2008; Kumar et al., 2010b). We have performed experimental infection of APMV-3 strains Wisconsin and Netherlands in 9-day-old embryonated chicken eggs, 1-day-old chicks and turkeys, and 2-week-old chickens and turkeys in order to investigate their tropism and pathogenicity. Birds were infected by the oculonasal route and the viral tropism and replication efficiency were evaluated by quantitative virology and immunohistochemistry of a wide range of possible target organs. In addition, a separate group of 1-day old chicks were infected intracerebrally to evaluate the potential of these viruses to replicate in

gastrointestinal tract, respiratory tract and pancreas (Warke et al., 2008).

misdiagnosis of APMV-3 as APMV-1.

neuronal tissue.

APMV-3 strains parakeet/Netherlands/449/75 and turkey/Wisconsin/68 (obtained from National Veterinary Service Laboratory, Ames, Iowa, USA) was propagated in 9-day-old specific pathogen free (SPF) embryonated chicken eggs via allantoic route of inoculation. The allantoic fluids from infected embryonated eggs were collected 96 h post-inoculation and virus titer was determined by hemagglutination (HA) assay with 0.5% chicken RBC. The virus titer in infected tissue samples was determined by the tissue culture infective dose (TCID50) method and by plaque assay in chicken embryo fibroblast (DF-1) cells (ATCC, Manassas, VA, USA) (Reed and Muench, 1938). For the plaque assay, DF-1 cell monolayers in 12 well plates were infected with different dilutions of tissue homogenates. The tissue homogenates were allowed to adsorb for 1 h at 37 °C, washed with phosphate-buffered saline (PBS), and then overlaid with 1 mL DMEM containing 0.8% (wt/vol) methylcellulose. The virus plaques in the DF-1 cell monolayer were visualized 96 h PI after staining with 1% crystal violet.

### **2.2 Pathogenicity index tests**

The pathogenicity of the APMV-3 strains Netherlands and Wisconsin was determined by two standard pathogenicity tests. These included the mean death time (MDT) in 9-day-old embryonated SPF chicken eggs and the intracerebral pathogenicity index (ICPI) in 1-day-old SPF chick (Alexander, 1998).

MDT value was determined following the standard procedure (Alexander, 1998). Briefly, a series of 10-fold (10−6 to 10−12) dilutions of fresh infective allantoic fluid in sterile PBS were made and 0.1 mL of each dilution was inoculated into the allantoic cavities of five 9-day-old embryonated SPF chicken eggs, which were then incubated at 37 °C. Each egg was examined three times daily for 7 days, and the times of embryo deaths were recorded. The minimum lethal dose is the highest virus dilution that caused death of all the embryos. MDT is the mean time in hours for the minimum lethal dose to kill all inoculated embryos. The MDT has been used to characterize the NDV pathotypes as follows: velogenic (less than 60 h), mesogenic (60 to 90 h), and lentogenic (more than 90 h) (Alexander, 1980b).

For determining ICPI value, 0.05 mL of 1:10 dilution of fresh infective allantoic fluid (28 HA units) of each virus was inoculated into groups of 10 one-day-old SPF chicks via the intracerebral route. The birds were observed for clinical symptoms and mortality every 8 h for a period of 8 days. At each observation, the birds were scored as follows: 0, healthy; 1, sick; and 2, dead. The ICPI is the mean score for all of the birds in the group over the 8-day period. Highly virulent NDV (velogenic) viruses give values approaching 2 and avirulent NDV (lentogenic) viruses give values close to 0 (Alexander, 1998).

#### **2.3 Pathogenesis assessment in chickens and turkeys**

1-day-old and 2-week-old SPF chickens (Charles River, North Franklin, Connecticut, USA) and 1-day-old and 2-week-old turkeys (Murry-McMurry hatchery, Webster City, Iowa, USA) were housed in positive pressure isolators in our BSL-2 facility. Birds confirmed to be negative for APMV-3 specific antibody by HI assay were further selected for pathogenesis

Pathogenicity of Avian Paramyxovirusserotype-3 in Chickens and Turkeys 591

turkeys. Gross examination of other organs including the brain, trachea, liver, kidney,

Fig. 1. Gross morphology of pancreas collected from 2-week-old turkeys infected with APMV-3 strain Netherlands (a) or Wisconsin (b): comparison with pancreas from uninfected

The brain, trachea, lung, spleen, kidney and pancreas showed the presence of viral antigen upon inoculation. Although APMV-3 strain Wisconsin replicated less efficiently in 2-weekold chickens, the titer of virus in lungs was somewhat higher than that of strain Netherlands, suggesting that it might be more respirotropic, whereas strain Netherlands

In general, infected turkeys had virus replication in fewer organs and for shorter duration than chickens of same age groups. Strain Netherlands was isolated from all of the tested organs except for the pancreas and kidneys in turkeys. Strain Wisconsin was isolated from all of the tested organs in turkeys. Results also suggested that turkeys are less susceptible to

All the chickens and turkeys infected with either strain seroconverted 14 days post infection confirming virus replication. Extensive immunofluorescence was observed in the brain, trachea, lung and pancreas, suggesting extensive virus replication in these organs (Kumar et al., 2010a). The study also suggested that the epithelial lining in the lungs and trachea are

APMVs are frequently isolated from wild and domestic birds around the world and have been placed into nine serotypes based on antigenic relatedness. Among these nine serotypes, APMV-1 (NDV) is the best studied because virulent NDV strains cause severe disease in chickens. APMV-2 to -9 are present in both free living and domestic birds, but

controls (left panels) reveals necrotic foci along the length of the infected organ.

infection with either APMV-3 strain compared to chickens (Kumar et al., 2010a).

appeared to be more neurotropic (Kumar et al., 2010a).

more privileged site for virus replication.

**4. Outcome of the study** 

spleen and lung showed normal tissue morphology with no noticeable gross lesions.

experiments and housed in negative pressure isolators. The 1-day-old and 2-week-old chickens and turkeys, in groups of 12 for each species and age, were infected with 0.1 mL (103 PFU) per bird of APMV-3 strain Netherlands or Wisconsin through the occulonasal route. Infections with the different strains were performed at separate times to avoid cross infections. An additional six birds of each species/age group remained as uninfected controls and sacrificed 14 day post infection (DPI) after collection of serum. Birds were provided with food and water *ad libitum* and monitored daily for any visible signs and symptoms twice daily. Three birds from each infected group were euthanized on 3, 5, 7 and 14 DPI by rapid asphyxiation in a CO2 chamber. The birds were swabbed orally and cloacally just before euthanasia. The following tissue samples were collected both for IHC and for virus isolation: brain, trachea, lung, spleen, kidney and pancreas. In addition serum samples were collected on day 14 when the three remaining birds in each group and the control birds were euthanized. Seroconversion was evaluated by HI assay (Alexander, 1980b).
