Multi-strain infection by infectious bronchitis variant viruses in broiler and breeder flocks in Latin America

Infectious bronchitis virus (IBV) causes a highly contagious respiratory tract disease in chickens, Infectious Bronchitis (IB), and some strains are nephropathogenic.  It is possibly the most economically important viral respiratory disease of chickens in regions where there is no highly pathogenic avian influenza virus or velogenic Newcastle disease (vND) virus and is found everywhere that broilers are commercially produced (5). And even in some countries endemic for vND (e.g., Peru and Colombia), IB is regarded as having similar or even higher economic impact than vND (12; present report). The virus can also cause significant egg production losses and mortality in commercial layers and breeders (17).

 

Coronaviruses, particularly IBV, are RNA viruses that present high genetic mutation rates. When those mutations occur in the spike gene they can result in the emergence of antigenic variant strain viruses or new serotypes (11,14). These variant strains are often partially or not controlled at all by live and inactivated IBV Massachusetts strains-based vaccines.

 

During the last eleven years, approximately, several countries in Latin America have reported an increasing incidence of clinical and subclinical outbreaks of Infectious Bronchitis (IB) caused by variant strains of the IBV (4,7,12,13,17).

 

This short article reports the findings in three broiler companies in Colombia (companies A, B and C; Midwest and northeast regions) which had several broiler breeders (five flocks investigated in company A) and broiler flocks (several flocks in companies B and C) presenting overt respiratory signs, nephritis, late mortality, decreased egg production and decreased fertile egg quality, investigated for IBV infection. Results from Elisa serology and a molecular diagnostic survey confirmed that flocks had been infected by either one or concomitantly by more than one of the following IBV variant strains: a) Q1 originally isolated in China (20), b) YE/L 2865/05 original molecular detection in swabs taken from industrial poultry in Yemen (19), c) K46/10 originally isolated in South Korea (10) and, d) PT/L 898/04 original molecular detection in swabs taken from industrial poultry in Portugal (19). Details of the clinical and diagnostic investigation and control attempts will be reported.

 

GD Animal Health. All molecular detections and genetic characterizations were carried out at the poultry diseases diagnostic laboratory GD Animal Health (Deventer, Holland). Samples were comprised by clinical tissues imprints from trachea, lungs, kidneys and cecal tonsils on FTA card (GE Healthcare Bio-Sciences Corporation, Piscataway, NJ, USA). The FTA card material was processed for IBV molecular detection (RT-PCR) and sequencing of PCR products as described elsewhere (16). In short, nine sample discs from the dried spot on each of the four FTA card circles were collected using a 2.0 mm diameter Harris micro punch and placed in a 1.5 mL tube. 270 µL of RNA Rapid Extraction solution (Ambion) was added to the tube and incubated for 5 minutes after mixing. Subsequently 200 µL of solution for RNA extraction (MagMAX system) was added. The RT-PCR was performed as described earlier (16). A fragment of about 350 base pairs of the S1 gene was amplified with the primers XCE1+ and XCE3- (3). The S1 amplicons were separated on a 1 % agarose gel, and visualized with ethidium bromide staining and an ultraviolet light transilluminator. The purified amplicon was sequenced (BaseClear, Leiden, The Netherlands) using both XC1+ and XCE3- primers. The sequence data were aligned using computer software (Bioedit, Ibis Biosciences, Carlsbad, USA).

 

When used, IBV serology was carried out locally (laboratories in Colombia) in commercial Elisa kits (Idexx® IBV Ab Test, Idexx® laboratories, Westbrook, Maine, USA).

 

Clinical signs, macroscopic lesions and flocks’ clinical and productivity data were collected when appropriate or possible for those flocks suspect of undergoing an Infectious Bronchitis outbreak.

 

In Table 1, it is presented the variant IBV strains diagnosed in the three poultry companies in Colombia. Four different IBV variant strains were detected (Q1, YE/L, K46/10 and PT/L ). The diagnosis of the Q1 and K46/10 strains was carried out in few broiler flocks in companies B and C respectively. Broiler flocks’ sampling and samples submission to molecular diagnostic were done at different occasions. Likewise, the detection of strains Q1, YE/L and PT/L in company A was done in five different breeder flocks sampled at different ages and sampling dates and samples submitted to the laboratory at a different occasions. Percentage nucleotide homology of the detected partial portion of the S1 spike gene in the variant IBVs on the FTA cards when compared with the originally GenBank-deposited sequences were 96.7-98% for the Q1 (accession numbers: AF286302 = original Q1 and HM446006 = Q1 from Chile) and, 96.3 % for YE/L (accession number = EF006524), 97% for the K46/L (accession number = JF804679) and 93% for the PT/L strain (accession number = EF066521). Further genetic comparison of the variant IBVs detected with the only IBV live vaccine strain (Massachusetts H120) available in Colombia indicated a quite small S1 protein gene homology between them (Table 2).

 

Table 1. Molecular detection of different IBV variants in Colombia in 2015.

 

Table 2. Percentage (%) nucleotide homology of a portion of the S1 spike gene in the variant IBVs on the FTA cards and the Massachusetts (Mass) H120 live vaccine strain used in all three companies (10,19,20; Dr. Remco Dijkman –  GD Animal Health, Deventer, The Netherlands – personal communication).

 

Elisa serology titers at slaughter in broiler flocks affected with the Q1 strain were quite high at company C (Maximum titer as high as 6000 and GMTs around 2500; vaccination scheme = 1 dose H120 spray at day one and one dose of Ma5 spray between 2-3 weeks of age) similar to what has been reported elsewhere (15). For company B, infected with the K46/10 strain, IB titers were at medium to normal levels (maximum GMT = 2769; vaccination scheme = 1 dose H120 spray at day one; average slaughter age = 36 days). In all investigated flocks’ serology for other respiratory disease (e.g., Newcastle, metapneumovirus infection) were not indicative of field infection.

 

Clinical signs in broilers were quite diverse in Company B and C and a more severe incidence of clinical disease was observed in those flocks infected by the K46/10 strain (Table 3). Late mortality (>4 weeks of age), renal and respiratory signs as well as septicemia due to secondary bacterial infection were quite common in affected flocks. In Company B affected by the K46/10 strain many infected flocks presented average mortality of up to 15% at slaughter age, most of it occurring during the last week of the grow out period (5th week).

 

Table 3. Relative incidence of clinical signs in broilers and breeder flocks infected with different strains of variant IBVs (incidence score: + = low / ++ = low to medium / +++ = medium / ++++ = high / +++++ = very high).

 

The investigated five breeder flocks in Company A were all clinically affected in a similar manner (Table 3). Severity of clinical signs varied from flock to flock but were always present and easily identifiable. Flocks with diagnosed infection by either the Q1 or the YE/L or the PT/L strain presented clinical disease at the ages of 27, 28, 30, 44 and 65 weeks (Table 1).

 

This is a quite unusual report of a multi-strain variant IBVs infection clinically and simultaneously occurring in a given country in Latin America. The Q1 strain has been causing severe losses to the broiler industry of several countries of South America during the last 7-8 years including Colombia (2,12,17).

 

For the first time in Latin America the Q1 strain was not only detected but also correlated with clinical disease in a breeder flock. In addition, three other variant strains never previously reported in Latin America (K46/10, YE/L and PT/L) were also detected and correlated with clinical disease and macroscopic lesions in broilers and breeders.

 

Company A, where Q1 and YE/L and PT/L variant strains were detected, has been seen recurrent clinical outbreaks in their breeder flocks, particularly during first 15-20 weeks of production although, in the present investigation, a 65-week flock was also found clinically affected by the YE/L strain. Of particular clinical severity (Table 3 for main clinical signs and lesions) was the infection of two young breeder flocks (27 and 28 wk of age) by the strain PRT/L. The general perception in the company is that this clinical situation from IBV infection in breeders is increasing in severity during the last several months even after each flock had been vaccinated with four live and four inactivated doses of IB vaccines during the rearing period (Mass strains only). Live Mass revaccination during production seems to slightly alleviate the clinical condition but never completely prevent/control it.

 

A very similar situation is occurring in broilers in Companies B and C which have had their broilers flocks challenged by the K46/10 and Q1 strains, respectively. During the mid-part of 2015 (July), an epidemiologically critical period in Company B, mortality of K46/10 affected broiler flocks averaged 14%. Broilers in that are vaccinated via spray at day one only with the H120 strain. The efforts for improving biosecurity and decreasing the environmental contamination plus a strong work for lessening mycoplasma vertical transmission to the broiler progeny in the company have improved considerably the IBV epidemiological situation in the company. By the end of 2015 the company had gone back to almost a normal clinical situation in the broiler farms by decreasing total mortality to an average of 5.5%. Clinical IB outbreaks in broilers caused by the Q1 strain in Company C are not new for this production system since the Q1 strain has been diagnosed in the company in the past.  Since the first diagnosis the company has been vaccinating broilers against IB at day one and in the field (2-3 wk of age) using two commercial Mass vaccine strains (H120 and Ma5). Nonetheless, the vaccination program has never been totally effective in controlling the Q1 strain. The same situation has been seen in recent past years in Chile, Argentina and Peru (12). A total control of the Q1 strain, at the level of both controlled protectotype trials and in the field, has only been accomplished when broilers where vaccinated with an association of a Mass vaccine strain with a 793/B-type vaccine strain (12,13,18). Such vaccination program cannot be used in Colombia at the moment since there is no 793/B-type live vaccine registered in the country. As for the K46/10 and YE/L strains there is no published information from protectotype trials indicating either the protection afforded by the Mass H120 strain alone or in combination with a 793/B-type live vaccine. From the S1 gene nucleotide homology between the K46/10, YE/L and PT/L strains with the Mass H120 strain depicted in Table 2, it can be concluded that the protection afforded by the Mass strains alone will most probably be quite low against these new variants circulating in the country just like it is against the Q1 strain (9,15). Some few years ago, Alvarado et al. (1,2) were able to carry out the first molecular detection of four unique IBV genotypes variants in Colombia although no subsequent work (pathotype and protectotype trials) has been done with those indigenous genotypes isolated from commercial layers and broilers.

 

Finally, at the moment, there is no epidemiological evidence/clue as to where from and how these IBV variants where able to arrive in Colombia and establish a new and quite economically damaging clinical picture in some broiler and breeder flocks of three different poultry companies. The same questioning has very often occurred in many parts of the world where new IBV variants have appeared that are genetically very similar to strains originally isolated/detected in another continent several thousands of kilometers away (7,17).

 

Such an unusual IB epidemiological situation in Colombia should be thoroughly investigated and the dissemination of the four IBV variants in the Colombian poultry industry clearly and quickly established. In addition, the K46/10, YE/L and PT/L strains should be isolated, pathotyped, protectotype trials carried out, and eventually, new and effective vaccination programs made available for the Colombian poultry industry.

 

 

 

 

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