Dave Cavanagh
Institute for Animal Health, Compton Laboratory, Newbury, Berkshire RG20
7NN, UK
E-mail: dave.cavanagh@bbsrc.ac.uk
Proceedings of the 24th Technical Turkey Conference p 31
Abstract
A coronavirus was shown to be involved with enteritis of turkeys in the
USA about 30 years ago. In young poults it has caused mortality upwards of 59%
whilst in older birds it is debilitating, resulting in underperformance with
regard to meat and egg production. During the last decade the coronavirus has
become one of two or more viruses associated in the USA with poult enteritis
and mortality syndrome (PEMS), equally devastating as the earlier coronaviral
enteritis. Even when very high mortality is not experienced, coronavirus
infection can result in reduced productivity, averaging about 5 US cents per
pound of meat. There is no vaccine; prevention is by sustained high levels of
hygiene, biosecurity and, preferably, single-age sites. Some confusion has surrounded
the identity of the causative coronavirus. Research in the 1980s and early
1990s suggested that the coronavirus from turkeys was virtually identical
genetically to the coronavirus of cattle, bovine coronavirus (BCoV). Later
research showed that several turkey isolates were genetically similar to
infectious bronchitis virus (IBV) of domestic fowl; IBV is very
different genetically from IBV. The genetically IBV-like turkey isolates were
not identical to each other; they differed from each other to an extent similar
to that between different serotypes of IBV. Immunofluorescence tests, using
tissues from turkey embryos or poults experimentally infected with genetically
IBV-like virus, have been used extensively in recent years in the US, revealing
that the virus is common in turkeys throughout
the country. The turkey coronaviruses cannot be grown in cell culture.
Laboratory diagnosis is currently being done by indirect immunofluorescence,
using infected tissues as substrate. However, commercially available ELISAs,
designed for detecting antibodies IBV, have recently been shown to be useful
for detecting infection of turkeys with the IBV-like coronaviruses. Turkey
coronaviral enteritis had only been described in North America until last year,
when molecular analysis proved that an IBV-like virus was present in the gut
contents, also observed by electron microscopy, of scouring turkeys in Britain.
The extent of coronavirus infection of turkeys in Britain and elsewhere beyond
North America is not known, but this discovery should provide the impetus to
examine it.
History
Turkey coronaviral enteritis was first described in the 1940s, in
Washington State, USA, although the causative agent was not identified until
about 20 years later. The
disease initially went under names such as ‘bluecomb disease’ and ‘mud
fever’. A devastating outbreak occurred in the major turkey producing state of
Minnesota in 1951, setting the scene for extensive research into the disease
(Nagaraja and Pomeroy, 1997). In the 1970s and 1980s it was the greatest
disease-related cause of economic loss in turkeys in Minnesota. Mortality can
be less than 10% but
as great as 50%, being greatest in very young birds. During the 1990s
attention was focussed on losses in poults attributed to ‘poult enteritis and
mortality syndrome’
(PEMS; Barnes and Guy, 1997). As the name suggests this was, by
definition, associated with poults up to 28 days of age. Although commonly
present at the time of PEMS, coronavirus is not the only pathogen that has been
associated with this disease. In particular an astrovirus has recently been
detected and shown to be able to reproduce PEMS-like disease in experiments
(Koci et al., 2000; Schultz-Cherry et al., 2000; Yu et
al., 2000a, b). Certain gut bacteria can exacerbate the consequences
of the viral infections (Guy et al., 2000). The co-incidence of coronavirus with PEMS is taken very
seriously in the US and monitoring the presence of the virus has been a major
part of attempts to minimise the effects of the disease. Steps that decrease
infection with coronavirus are likely to also reduce losses caused by other
pathogens.
Clinical manifestation
Turkeys affected by coronaviral enteritis huddle together, stop eating,
lose weight and have wet droppings, older birds exhibiting depression. These
signs may persist for up to two weeks and recovery of weight, if achieved, may
take several weeks. Laying birds experience a drop in production. Affected
birds (diarrhoea, dehydration and growth depression of 40% or more) between 7
and 28 days of age are considered to be experiencing PEMS. At its simplest this
is defined as deaths exceeding 2% in this period. The most severe form is
called spiking mortality syndrome of turkeys (SMT), in which mortality is
greater than 9% in the 7 to 28 day of age period, including three or more
consecutive days in which mortality is 1% or greater. Mortality of 59% has been
reported. In a workshop organised by Roche in the USA in 1998 (Clark and
Vaillancourt, 1998), one veterinarian described outbreaks in Texas, 1998, in
which on one farm mortality had reached 29-31% in two houses by six weeks of
age. On another farm mortality had reached 26 to 49% in three houses; the
remaining birds were destroyed. A less severe form of PEMS is called excess
mortality syndrome (EMS) in which mortality of 1% for three consecutive days is
never reached. Both EMS and SMT may be experienced on the same farm,
perhaps indicative that other factors
can exacerbate the viral infection.
Environmental features
In the States there is seasonality with respect to both the incidence of PEMS and of the coronavirus,
centring on October. Whilst there is no direct correlation with heat or
rainfall separately, the incidence of disease follows months of high humidity.
The fly population rises in such circumstances; they may spread the virus in a
mechanical way. One company has observed that a case of PEMS experienced in
Spring in Carolina will not spread, unlike a case in July to September. That
might reflect the incidence of flies
amongst other things. The coronavirus can persist in turkeys for several months
and may be excreted for months after initial infection.
Economic loss
Devastating outbreaks of PEMS, in which 20 to 40% or more of poults may
be lost in a few weeks, is clearly a cause of serious economic loss. Many
instances are not as dramatic but performance is below par to the extent that
average losses, over many crops, in coronavirus positive flocks have been
approximately 5 cents per pound of meat. Losses have been attributed to a decrease
in livability from about 93% to 86% on average, poor feed conversion, poor
weight gain and final weights and increased medication. Where measures were not
taken the losses increased with successive flocks.
Action
There is no doubt that in the US improved performance has started by
increased monitoring of flocks for the presence of the coronavirus. This has
enabled action to be focused on farms where incidence of the virus is high and
has provided one marker, in addition to increased performance, to assess the
success of preventive measures. The latter do not involve vaccines; there is no
vaccine against the virus.
Measures taken, therefore, have involved changes in rearing practice and
improved biosecurity. Delegates at the 1998 workshop reported that switching
from multi-age to single age sites (brood and move or all-in- all-out) was a
very important element in reducing the incidence of disease. Co-incident with
this step were better litter management and other aspects of biosecurity: care
regarding the disposal of dead birds; minimising motorized traffic onto sites;
disinfection of vehicles; disinfection of
footwear. Given that the virus is excreted in large amounts in faeces
any creatures, in addition to humans, can spread the disease. Other domestic
animals and pets should be kept well away from turkey houses, which should be
made inaccessible to wild birds. Vermin such as rats and mice are also a high
risk factor. Bovine coronavirus (BCoV) has been shown unequivocally, by
experiment, to be able to cause disease in
turkeys (Ismail et al., 2001). Therefore cattle should be kept well away from turkeys and
strict disease security measures observed when trafficking between the two
species.
As might be expected, an increase in downtime between flocks has proved
beneficial. Downtimes of less than 10 days were insufficient and three to four
weeks were worthwhile in turning a site around. The incidence of other
pathogens was also reduced by this and the other measures taken.
Not one coronavirus but two?
During the 1980s serological analysis indicated that the turkey
coronavirus isolates of the time were closely related to BCoV. This link was
strengthened when it was subsequently shown that some of the genes of the
turkey viruses were 99% the same as BCoV. Such a high degree of identity is
suggestive that the turkey viruses were indeed BCoVs. This view of the identity
of the turkey coronavirus went unchallenged until the late 1990s when research
at the University of North Carolina showed that some isolates of coronaviruses
from turkeys were actually genetically
related to infectious bronchitis virus (IBV) of domestic fowl (reviewed
by Guy, 2000; Breslin et al., 1999a, b). The sequences of the genes of the IBV-like turkey viruses
differed from chicken coronaviruses by about 10%. The turkey gene
sequences had never been seen in any IBV (chicken) isolates, strengthening the
view that bone fide
turkey viruses had been sequenced. Last year we discovered a coronavirus
in turkeys in Britain, the first unequivocal demonstration of such outside
North America. The virus came from two-week-old poults that were on a multi-age
farm. The birds had fluid caecal and intestinal contents, 20% of them showed
stunting, unevenness and lameness; 4% died. Electron microscope analysis at the
Veterinary Laboratories Agency central veterinary laboratory, Weybridge, of gut
contents revealed
particles that looked like a coronavirus. Given the American finding
that coronaviruses of turkeys are genetically like IBV, Dick Gough sent
material to us at Compton, as we had been using molecular techniques to study
IBV for many years. IBV exists as a great many serotypes – many dozen, at
least. Serotypes differ by about 10% in several genes (and by far more in the
gene that encodes the large surface protein). We have developed polymerase
chain reaction (PCR) procedures to detect many, if not all, types of IBV; the
end of the genome is particularly strongly conserved (Dalton et al., 2001). Our first PCR indicated that a coronavirus
– an IBV-like virus – was indeed present in the guts of the British turkeys. We
did several additional PCRs to amplify two genes that are possessed by IBVs but
not by
mammalian coronaviruses, such as BCoV; the turkey virus had these two
genes. Furthermore, the sequences of these two genes differed from their
counterparts in IBVs by about 10% (Cavanagh et
al., 2001a). Thus a coronavirus is present in turkeys
beyond North America. The extent of this presence in Britain and elsewhere is
completely unknown. It follows that we have no idea of the economic importance
of coronaviruses in turkeys outside the USA and Canada. The British turkey
coronavirus was about 10% different from the three American ones that had been
sequenced – and which differed from each other by about 10%. This raises the
possibility that the IBV-like coronaviruses in turkeys might exhibit a degree
of genetic heterogeneity similar to that of IBVs in domestic fowl. The
accumulated evidence of the past few years suggests that the genetically
IBV-like viruses from turkeys are the ‘real’ turkey coronavirus.
Notwithstanding, the recent demonstration that experimental infection of
turkeys with BCoV can result in disease (Ismail et
al., 2001) indicates that BCoV is a potential threat to
turkeys.
Biological differences between turkey and fowl coronaviruses
Although the turkey coronaviruses have gene sequences different from
those of IBVs they have not, according to research to-date, got any distinctive
genetic characteristic. Notwithstanding,
fowl IBVs and turkey IBV-like coronaviruses have different biological
characteristics. IBVs grow primarily in the respiratory tract of fowl, mostly cause
respiratory disease and grow readily in the chorioallantoic cavity of domestic
fowl eggs. In contrast, the turkey coronaviruses grow in the alimentary tract, including
the bursa of Fabricius, cause enteritis and cannot be grown in the
chorioallantoic cavity of fowl, or turkey, eggs. Some experiments have been performed in the US to see if the turkey
coronaviruses can cause disease in chickens. In one laboratory domestic fowl
chicks were inoculated with an IBV-like turkey coronavirus; the virus grew but
without causing any disease (Guy et al., 1999). In another laboratory not only was no disease caused but also there
was no evidence of the turkey virus having replicated in chickens (Ismail et al., 2001). So, biologically TcoV and IBV are
distinct. I believe that coronaviruses will be isolated from many
avian species in the future and that many of them will have close
genetic similarity to IBV, although the species specificity of the viruses and
other biological characteristics might be different. In this regard we have recently
shown that coronaviruses present in pheasants are genetically close to IBVs, to
a similar extent as the turkey and chicken viruses (Cavanagh et al., 2001b). Moreover, the pheasant isolates
exhibit heterogeneity amongst themselves to the same extent as do IBVs amongst
each
other. Interestingly the pheasant viruses can be grown in the
chorioallantoic cavity of fowl eggs. In a single experiment fowl chicks
inoculated with a pheasant isolate
did not develop disease (R. E. Gough, personal communication). It should
be borne in mind that fowl IBVs do grow, to some extent, in alimentary tract
tissues of fowl, albeit asymptomatically (Cavanagh and Naqi, 1997). Many, perhaps
all, strains will grow to some extent in kidney, with some strains being highly
nephropathogenic. Pheasant viruses have been associated with nephritis in
pheasants.
There is probably a wide spectrum of avian coronaviruses that are
genetically close to IBV. A better analogy might be a sphere full of glass
beads (representing coronaviruses), the beads being in groups of different
colours (each colour representing a coronavirus from a different species of
bird) but with the colours shading into each other at the imprecise periphery
of each group of beads (viruses at the peripheries having some biological
characteristics shared with the viruses on each side). In 1987 it was reported
that application of an IBV ELISA in Israel revealed the presence of ‘IBV’
antibodies in turkeys exhibiting rhinotracheitis (Weisman et al., 1987). This phenomenon has been repeated
in the States, where a commercially available IBV ELISA has been used to detect
infection of turkeys in the field by IBV-like coronaviruses (Loa et al., 2000). (A goat anti-turkey
immunoglobulin conjugate was used in place of the anti-chicken immunoglobulin
conjugate). This would seem to be a good approach to use to start an assessment
of coronaviruses in turkeys beyond North America.
Acknowledgements
This work was supported by the Ministry of Agriculture,
Fisheries and Food, Great Britain.
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