I. Barbieri 2
L. Gavazzi3
G. Tosi2
1 Ex, Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Sezione di Microbiologia e Immunologia, Università degli Studi di Milano, via Celoria 10, 20133 Milano, Italy
2 Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna, via Bianchi 7, 25120 Brescia, Italy
3 Amadori Group, Cesena, Forlì, Italy
Summary
The authors describe the isolation and characterization of IBV isolates from Italian poultry farms. These isolates came from different chicken breeds (pullets, broilers) using different vaccination schedules.
The isolates, of which the AZ-40/05 has been the most studied, showed identical patterns of virus-neutralization to mono-specific antisera from each other, but they are distinct from the usual vaccine strains and from numerous other previous isolates in US, Europe and Australia. Sequence analysis of a portion of the S1 spike gene likewise suggested that these isolates were also distinct from the previous, but showed some degree of homology with recently described isolates from China. Another interesting aspect is that such isolates adapted very soon (2 passages) to embryonated egg, reaching very high titres (>10 7.5 EID50 /0.1 ml) in 6-7 passages, with uncommon very severe lesions in the embryo.
For some decades, the appearance has been recorded of ever-new serotypes or variants of infectious bronchitis virus (IBV) in Italy, as well as in the rest of Europe and in other Continents. Up to now, over 60 different serotypes of virus worldwide (at least 13 in Italy) have been reported (7, 9, 16, 17). Most of them have been sporadic isolations, but sometimes their frequency has been quite high, persistent and widespread, as in case of Massachusetts, Australia T, Arkansas DPI, AZ-23/74, D-274, 793 B, AZ-27/98 and some other strains (5, 14, 17). The isolates, which spread, in these last few years, to many European countries and have often been reported as "It-02" variant (12), seem to belong antigenically, or are very similar, to the AZ-27/98 serotype, isolated in Italy since 1997-98 (15, 17).
The main reason for such antigenic instability in IBV would derive from the particular mechanism of coronavirus replication, which occurs by discontinuous transcription of RNA (6 mRNAs), with changes in nucleotide and, consequently, in amino acid sequences of the S1 part of the virus spike protein (3). It is believed that, if a cell becomes infected by two different virus particles, new recombinants may be generated. However, the possibility of spontaneous appearance of a latently pre-existent serotype cannot be excluded. Biomolecular research has shown that a new serotype can emerge as a result of just a few changes in amino acid composition of the S1 spike protein, with the majority of the viral genome remaining unchanged (4).
The tropism of the different strains of virus is also becoming broader and more variable: respiratory, urogenital, gastrointestinal or mixed, both in layers and in broilers. Moreover, coronaviruses of the same group, to which IBV belongs (3rd group), have been isolated from other wild and domestic birds; they are currently being studied (5).
The objective of the present paper is to report on certain recent isolations of IBV from chickens with severe kidney lesions, seeming to be similar or identical to each other, but apparently different from the other main serotypes, already known and widespread in Italy and throughout the world.
Materials and methods
Clinical outbreaks. Light six-week-old pullets of three different breeds, reared in separate houses of the same farm, vaccinated as one-day-old with Mass H120. Broilers of the same breed, reared in three separate farms, five-wk-old, previously vaccinated as one-day-old with Mass H120 and as 18-20-day-old with UK-4/91.
Substrate. 9/11-day-old specific pathogen free (SPF) embryonated chicken eggs were used.
Virus isolation. Samples of kidney tissue, from chickens showing lesions, were homogenized in phosphate buffered saline (1:5 w/v), treated with antibiotics and inoculated into the allantoic sac of 10 embryonated eggs. After incubation at 37°C for 8 days, the eggs were daily candled and examined for specific IBV lesions (15). Allantoic fluids from a number of eggs were harvested 48-72 hr post- inoculation. Eight serial passages of the virus were performed before to do the cross-neutralization tests. The isolates were identified as AZ-40/05, 1730/05 and 1732/05.
IBV strains. In addition to the mentioned isolates, particularly AZ-40/05, also the Mass.41, AZ-23/74, Arkansas DPI, UK-4/91 and AZ-27/98 serotypes, as well as another recent Italian isolate, the FO 7856-05, were used.
IBV monospecific antisera. A panel of 14 specific polyclonal antisera was used: AZ-40/05 (new isolate), AZ-27/98, FO-4682/99 (It -02), AZ-23/74, Mass.41, Arkansas DPI, Gray, Australia T, UK-4/91 (793 B), AZ-446/66, 624I, PV-1731/65 and BS-216/01 (15). All sera were raised in 5 to 6-wk-old SPF chickens, kept in isolation units, inoculated twice at interval of 21 days and bled 2 wk later; the sera were filtered through Millipore 0.22 μm membranes, inactivated at 56°C for 30 min, lyophilized and stored at -20°C (15).
Virus-Neutralization test. To establish the possible antigenic correlation of new field isolates with some of the most common European, American, and Australian IBV serotypes, VN tests were performed in embryonated eggs, according to the very sensitive method variant virus−constant serum (log10 virus dilution and 1:5 serum dilution). Virus and serum were kept in contact for 1 hour, then the mixture was inoculated in eggs (five eggs for dilution). The embryos were candled and examined for specific lesions within 8 days. The neutralization indexes (NIs) were calculated according to the well-known method of Reed and Muench. Only values of NI> 2 log10 were considered positive. A negative serum was always included in the tests.
Molecular characterization. Reverse transcription-polymerase chain reaction ( RT-PCR) test was performed according to described methods (4, 6) on allantoic fluids, using primers HYBRIDIZE that can recognize the following most common European IBV types: Mass.41, 793B and FO-4282/99 ( It 02). RNA was extracted from allantoic fluids and purified by the method of Chomezynski and Sacchi (6), and stored at −20°C.
RNA was detected by RT-PCR assay, with XCE1+ and XCE3– primers, which are able to amplify a fragment of 383 bp of hypervariable region from nucleotide 705 to 1086, common to all IBV types. Then, a nested PCR was performed combining XCE3– with BCE1+ and MCE1+ primers, specific respectively for 793B and Mass.41 types, able to amplify cDNA fragments respectively of 154 and 295 bp (4). In addition a nested PCR was performed using It-O2F and It-O2R primers specific for FO-4682/99 (It-O2) strain, are able to amplify a cDNA fragment of 330 bp. The position of nucleotides, where primers hybridize with gene sequence, was XCE1+ =728-749; XCE3– =1093-1112, BCE1+ = 958-978; MCE1+= 817-837; It-02F=832-852; It-02R=951-970. The amplified fragments were analyzed by 1.7% agarose gel-electrophoresis, stained with ethidium bromide and observed with an ultraviolet transilluminator. A standard molecular weight was added and used to determine the size of the cDNA fragments.
Direct sequencing of various strains was performed on PCR products after gel purification with Qiaquick gel extaction kit (QIAGEN) with primers XCE1+ and XCE3– (ref. 15) by means of a Big Dye Terminator DNA Sequencing Kit on ABI Prism 310 Automatic Sequencer (Applied Biosystem, Foster City, CA).
Comparative analysis of nucleotide and deduced amino acid sequences was carried out with ClustalW Package of DNAStar (MegAlign v.5, 2001; DNAStar inc., Madison, WI). Sequences were available with Gene Bank accession no. EF186232 and EF186233.
Results
Clinical outbreaks. The flocks of pullets showed mild respiratory and enteric symptoms, but very severe kidney lesions, with mortality varying from 0.2 to 1.0 % over two weeks in the three different houses with three different breeds (AZ-40/05 isolate). The flocks of broilers showed depression, respiratory and enteric symptoms with wet litter, very severe kidney lesions and a mortality varying from 0.5% to 3.5% (1730/05, 1732/05 and FO-70856/05 isolates).
VN test. The AZ-40/05, 1730/05, 1732/05 and FO-70856/05 isolates showed neutralization (NI> 6.5 log10) by antiserum obtained from field convalescent birds infected with AZ-40/05 isolate and from SPF chickens, twice inoculated with 8th passage of such a virus. While, they were not neutralized (NI<2.0 log10) by any other antisera against many previously reported serotypes. Conversely, the AZ-40/05 antiserum did not neutralize any other better-known IBV serotype. However, the complete results are reported in Table 1.
RT-PCR test and sequence analysis. RT-PCR tests showed that the AZ-40/05 isolate seems to be related to Chinese QX isolate, but unrelated to the most common European IBV types 793B, AZ-27/98, FO-4882/99 (It-02) and Mass.41.
Nucleotide sequence analysis of partial genomic S1 region (383 nucleotides) revealed that AZ-40/05 showed 97.9% homology with QX isolate; as far as it concerns amino acid sequence, it showed no substitution compared to QX isolate (100% homology).
From the nucleotide and amino acid sequence analysis, the AZ-40/05 isolate showed a very high homology (100%) with the QX Chinese reference strain (10), against only 86% to 77% with other IBV serotypes previously reported. Anyway, the complete results of amino acid sequence of S1 protein of six different strains of virus are reported in Figure 1.
Discussion
A new serotype of IBV was isolated during 2005 in some poultry intensive areas of Italy (1,18). As regards our isolates, AZ-40/05, 1730/05, 1732/05, FO-70856/05 serologically identical, they differ completely from the serotypes or variants reported prior to 2004 in Europe and from some of the most common serotypes isolated in the US and in Australia. However, very few are known as regards the most recent isolates in Asia. Most recent isolates in Italy, of which the AZ-40/05 is representative, seem to be related, from the molecular point of view (100% amino acid homology), to the Chinese QX strain (9), even if it regards a short fragment (383 nucleotides) of the considered S1 spike protein; while, with regard to the most common European serotypes, the homology is about 86% and only 77% for the Massachusetts serotype. Similar strains have been isolated in some other European countries, with the first appearance, as D-388 strain, in The Netherlands in 2004 (8, 13). Nevertheless, it seems to be a little rash to consider them as a strain coming from China, on the grounds of the genetic characteristics of only a short part of the spike, as it has been thought and also reported (1). So, it would seem more appropriate to consider them as QX-like strains. So far, serological tests have not yet been reported for the various QX-like isolates.
All four strains, isolated in our laboratories, adapted unusually quickly (two passages) to embryonated eggs, inducing classic, but very severe lesions to the embryo (stunting, dwarfing, curling, mesonephros persistence) and reaching very rapidly high titres (> 7,5 log10/0,1 ml) after 6-8 passages (18). Such a situation never occurred, at least regarding our previous isolations, except in cases of vaccinal strains, as occurred with many isolates of Dutch variants D-274 and D-1466 and the English strain UK-4/91, after their introduction to Italy as vaccines (17). This rapid adaptation to embryo could lead also to consider a possible recombination between a vaccinal strain, well adapted to embryo, and another pre-existing wild strain.
Further investigations, which should be extended to different European countries, could allow the origin and spread of this new serotype to be determined, together with its exact relationship to the Chinese QX strain. On this matter, an interesting research, carried out in Quebec (Canada), showed that certain IBV isolates, which bound two different monoclonal antibodies, considered specific to the Massachusetts and Arkansas serotypes, after sequencing showed a part of the S1 spike protein gene, which was typical of the Massachusetts serotype, whereas the remainder was similar to the Arkansas DPI serotype. Because the birds, where from the virus strain was isolated, had been vaccinated with the Massachusetts strain, a recombination could have occurred between the two viruses (11). A mosaic of Mass+B1648+793B sequences was also reported in Hungary, revealing a potential field recombination (2). It follows that sequencing of various isolates should be broadened, in order to better define the right genotype of the virus and its eventual origin and relationship to other types.
More detailed molecular biology and immunological studies lie ahead in various laboratories. Nevertheless, broader cooperation and comparison of work between different laboratories would be appropriate and desirable to obtain surer results and draw more accurate and unequivocal conclusions on such important and, as regards certain aspects, complex infectious disease.
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Paper presented at the 44th Congress of the Italian Branch of World Veterinary Poultry Association, Forlì, 29/30 September 2005
