Infectious Coryza is a highly contagious bacterial disease caused by Avibacterium paragallinarum (formerly called Haemophilus paragallinarum). It often affects the upper respiratory tract of chickens (Gallus gallus) but has been also described in quails and parrots. Birds of all ages are susceptible. The disease is well known in laying hens although often goes unnoticed in subclinically infected broilers due to difficulties of the standard bacteriological methods. On the other hand, it is important to differentiate infectious coryza caused by Avibacterium paragallinarum from “coryza of turkeys", which is completely different and is caused by Bordetella avium.
The disease has worldwide distribution and it is mainly found in countries with intensive production where overcrowding and stress increase susceptibility of birds. Economic losses are produced due to a significant reduction in egg production (up to 40%) in layers or breeders after reaching peak production; weight loss in broilers (caused by diarrhoea and reduced consumption of water and food); and increased number of discarded chickens either at slaughter due to suffocation before being hung on the hook or due to dermatitis and cellulitis.
Pathogenicity and virulence of Avibacterium paragallinarum
The haemagglutinins (antigens that produce haemagglutination of red cells) of Avibacterium paragallinarum are structures mainly related to antigenicity, pathogenicity and immunogenicity. These haemagglutinins are antigens that agglutinate chicken red blood cells. In fact, these are adhesins that allow attachment of bacteria to structures on the mucosa cell surface and are considered essential for the first stage of infection. The chicken red blood cells share the same superficial receptors of the chicken epithelial cells of the respiratory tract mucosa. Thus, the active protection of an infected bird mainly depends on the presence of humoral antibodies, which inhibit the adhesion by neutralizing the hemagglutinin action. Other virulence factors, including the capsule in strains that cause septicaemia, could act by inhibiting phagocytosis and allowing distribution of the Avibacterium paragallinarum in other tissues.
Symptoms and lesions
Clinical signs include: nasal discharge, facial swelling with noticeable swelling of the face, lacrimation, anorexia, diarrhoea and it can also be hearing a snore or tracheal rattle when the lower respiratory tract is affected. Inflammation of the wattles is very rare but can occur in roosters.
According to clinical signs and head injuries, sick birds may be classified into four grades: Grade 1 (mild conjunctivitis); Grade 2 (periorbital swelling and sinus with or without conjunctivitis or tearing); Grade 3 (oedema and swelling of periorbital area and sinus, sinusitis, nasal and eye discharge, partial closure of the eye); Grade 4 (conjunctivitis with abundant nasal and eye discharge, severe periorbital swelling and sinus area, eyelids stuck and complete closure of eyes). Only those chickens having grades 2, 3 and 4 (Photos 1, 2 and 3) are considered to be sick.
Photo 1: Grades in chickens experimentally inoculated and observed at the 2nd day post-infection.
A. Grade 0 (lower chicken) without any lesion and Grade 1 (top chicken) with mild conjunctivitis.
B. Grade 2. Conjunctivitis with partial closure of the eye and swelling of periorbital area and paranasal sinuses.
C. Grade 3. Conjunctivitis, eyelids closed but unbounded and obvious swelling of periorbital area and paranasal sinuses.
D. Grade 4. Conjunctivitis with eye completely closed, eyelids stuck and very severe swelling of the periorbital and sinus area.
Photos obtained from: SORIANO VARAS, E.; TERZOLO, H.R. Epizootiología, prevención y control de coriza infecciosa. Epizootiology, prevention and control of infectious coryza. Veterinaria México 35 (3): 272, 2004.
Photo 2. Infectious coryza in natural infection of hens. A. Grade 2. Note the slight swelling of the infraorbital sinus and abundant thick mucus when opening the peak. B. Grade 4. Note the severe swelling of the face, eyes completely closed with lids bounded, nasal and eye secretions and abundant mucus into the oral cavity.
Photo 3. Infectious coryza Grade 4 in a 35 day-old broiler experimentally inoculated and observed at the 2nd day post-infection. Note the severe swelling of the face, the abundant eye discharge with eyes completely closed and attached lids due to an intense inflammation.
When Infectious coryza is associated with other viral or bacterial infectious agents such as infectious bronchitis virus, Mycoplasma gallisepticum, Avibacterium gallinarum (previously named Pasteurella gallinarum), Escherichia coli, Salmonella spp. or Pasteurella multocida, the disease worsens and prolongs its course and is denominated “Complicated Infectious Coryza " (Figure 1).
Figure 1. Comparison between the expected standard production of Lohmann Brown Classic line and the real production observed during a severe outbreak of Complicated Infectious Coryza associated with Mycoplasma gallisepticum and intestinal parasites in addition to bad farm management and poor biosecurity conditions. Note that the viability of hens is almost unaffected while the lay of eggs significantly decreased. Note the sharp fall of egg production from week 32 and the difficulty of maintaining it after successive antibiotic treatments (weeks 33, 34 and 35). On week 46 the food supply was suspended for 5 days to produce molting together with total absence of production, which restarted from week 51 onwards.
In broilers, Avibacterium paragallinarum may cause “swollen head syndrome" in which it is associated with other infectious agents, such as Escherichia coli, Bordetella avium, Mycoplasma gallisepticum and/or turkey rhinotracheitis (TRT) virus or infectious bronchitis virus, among others.
Poultry that suffer Complicated Infectious Coryza are not easily cured and usually the affected birds remain with several sequels, being common an increase in mortality and many discarded birds.
Duration of illness
When Avibacterium paragallinarum is not associated with other infectious agents, it has a short-term incubation period of 1 to 3 days and the symptoms persist for 3 to 7 days. In these uncomplicated cases, the disease is generally characterized by high morbidity and low mortality although it can also be asymptomatic without any mortality, or produce septicaemia and death, according to the virulence of the acting strain. However, when other bacterial or viral agents are associated with Avibacterium paragallinarum, the disease continues for several weeks (Figure 1).
Routes of infection and persistence of the pathogen in farms
The bacterium does not persist in the environment for a long period of time and therefore, the main reservoir of infection is the same sick birds. After recovering from the disease they continue to harbour Avibacterium paragallinarum but without showing any sign of disease. Despite these carrier birds remain apparently healthy, they are able to infect new susceptible young birds that are introduced into the farm. Hence, multi-age farms are not recommended.
In addition, Avibacterium paragallinarum can be introduced into free farms by air. For these reasons, biosecurity measures and proper distance between farms are very important to prevent entry of the pathogen, since once present on premises, eradication is very difficult, especially in multi-age farms.
The clinical diagnosis of classical infectious coryza is relatively easy when it is not complicated with other infectious agents. However, even in these cases, it is very important to send samples to the laboratory to confirm the diagnosis and determine with certainty the acting serogroup and preserve strains that eventually might be used in vaccines adapted to the affected zone.
When clinical signs of infectious coryza are observed, it is recommended to send three to five heads to a diagnostic laboratory. These heads should be selected from birds recently suffering from acute signs of the disease. It is important that heads are shipped frozen so that they remain as such throughout transportation until arrival at the laboratory.
Bacterial isolation is performed from mucus samples taken from the infraorbital sinus. This requires cauterizing the skin covering the infraorbital region using a red hot metal spatula heated with a Bunsen burner. Afterwards, a skin incision is practiced onto the cauterized area, the skin is separated and a sterile swab (Photo 4), previously moistened with sterile nutrient broth or phosphate buffered solution at slightly alkaline pH (7.2-7.4), is introduced through the incision into the infraorbital sinus. Another sampling technique involves cutting the upper beak behind the nostrils followed by the introduction of a sterile moistened swab, as mentioned above, to collect the mucus directly from inside the nasal turbinates. Additionally, in septicaemic cases, it is possible to isolate this bacterium from internal organs, such as lungs, air sacs, livers and spleens; in acute cases Avibacterium paragallinarum has been isolated even from inside eyeballs. Heads and organs may be transported frozen using dry ice.
Photo 4. Swabbing the inside of the infraorbital sinus of a broiler. Prior to cutting the skin was cauterized to prevent contamination of the sample.
Isolation of Avibacterium paragallinarum
To be able to isolate this bacterium it is necessary to provide the intracellular coenzyme Nicotinamide Adenine Dinucleotide (NAD or V Factor) to the culture media, except for some independent strains that do not require free NAD in the culture medium.
Swab samples, taken from the infraorbital sinus can be streaked onto any rich nutrient agar plates (i.e. Columbia or Brain Heart Infusion) added with 7 per cent of whole desfibrinated (unhemolysed) blood previously seeded with a nurse strain, for instance Staphylococcus aureus. Staphylococcus aureus colonies haemolyses erythrocytes liberating free NAD, which diffuses to the nearby agar allowing a faint growth of Avibacterium paragallinarum. Hence, after incubation Avibacterium paragallinarum will grow as very small colonies surrounding the nurse strain and only over its haemolytic zone (Photo 5).
Photo 5. Development of Avibacterium paragallinarum on Columbia agar with addition of 7% (v/v) non-haemolysed bovine blood after 2 days of incubation period at 37°C. Avibacterium paragallinarum only develops around the colonies of Staphylococcus aureus (satellitism) and within the zone of hemolysis (arrows).
Alternatively, or additionally, the swab may be streaked on plates of any of the above mentioned rich nutrient agars adding 5 to 7 per cent of desfibrinated haemolysed horse blood. The blood may be haemolysed by heating at 56°C for 30-40 minutes into a water bath and afterwards added to the agar. Other preparation method is to haemolyse the blood by pouring it into a previously heated agar at 56°C into a water bath (chocolate agar). The heating process releases intracellular NAD from the erythrocytes and at the same time inactivates the growth inhibitors of the blood. Agars prepared with haemolysed blood do not require addition of the afore mentioned nurse strain. Besides, a selective agar may be prepared by adding bacitracin (5 IU/mL); cloxacillin (5 μg/mL) and vancomycin (20 μg/mL). In these selective or non-selective agars Avibacterium paragallinarum will grow as bright mucoid colonies (Photo 6).
Photo 6. Abundant development of Avibacterium paragallinarum colonies in pure culture after 2 days of incubation period at 37°C onto agar Columbia with addition of 7% (v/v) hemolyzed horse blood. The haemolysis was produced by heating the blood into a water bath at 56°C for 35 minutes. Avibacterium paragallinarum develops across the whole plate as bright mucoid colonies.
Incubation is performed at 37 °C for 48 hours under a microaerophilic atmosphere obtainable by any of these procedures: the classical candle jar method; any CO2 incubator; replacement of 10 per cent of the air contained in a jar by carbon dioxide; or any commercial gas mixture for generating Campylobacter microaerophilic atmospheres.
As can be seen in Photo 5, using the unhemolysed blood agar Avibacterium paragallinarum grows very weakly and only around the nursing colonies (phenomenon called satelitism). Instead, as shown in Photo 6, using agar with the addition of lacquered or lysed blood, Avibacterium paragallinarum grows as large colonies covering the entire agar plate without depending on the feeding strain.
Isolates may be kept frozen in liquid nitrogen or at -80ºC by depositing (without disaggregation) a lump collected from the growth of a plate into a small 1-2 mL vial containing 0.5 mL of tryptose phosphate broth or brain-heart infusion added with 5 per cent (v/v) of decomplemented equine serum and 17 per cent (v/v) of tyndallized glycerol.
Serogroups, serovars and serological tests
According to the Page scheme, Avibacterium paragallinarum is classified into three serogroups, designated A, B and C. This serotyping is based on a haemagglutination inhibition (HI) test using rabbit antisera against A, B and C standard strains, fixed chicken red cells and the haemagglutinins of the strain to be classified. This HI test allows knowing which serogroup is present in an outbreak and could help make rapid decisions regarding which vaccine should be given.
The Kume scheme modified by Blackall recognised nine serovars distributed into the three Page serogroups: A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3 and C-4. This test is used to determine the circulating serovars and, therefore, may be used to define which serovar/s should be included in the bacterins administered in a particular region.
Several serological tests for detection of Avibacterium paragallinarum antibodies in chickens have been described: hemagglutination inhibition (HI), gel precipitation, agglutination, latex agglutination and ELISA.
Currently inactivated bivalent (serovars A-1 and C-1) or trivalent (A-1, B-1 and C-2) bacterins are used according to the incidence of serovars in different geographical regions. However, there have been described cases in which vaccines did not adequately protect, as happened with outbreaks caused by “variant” serovar B strains, which have very high pathogenicity. These B strains had to be added to the standard vaccine formulation marketed in some regions where they are present. Vaccines based on killed antigens have no cross protection between the three serogroups, so it is necessary to include those serogroups that are present in the geographical area to which the vaccine is intended to be used. Furthermore, challenge tests carried out between vaccinated and unvaccinated chickens showed that some serovars of the same serogroup (either B or C) have no cross protection among themselves. Therefore, in certain cases, application of autogenous bacterins or auto-vaccines, which include the strain present in the farm, is an effective preventive measure.
Vaccines are injected to the birds during rearing, usually before posture, and it is recommended to vaccinate subcutaneously into the dorsal region of the neck, in the distal area of the head, although vaccines can also be intramuscularly inoculated into the breast. Immunization of birds should be performed before 20 weeks of age, administering two doses separated by an interval of 3-4 weeks, considering that the last dose has to be administered about 3 weeks before hens start laying. On the other hand, it is recommended to revaccinate 10 days after completing the molting. In areas exposed to the disease, it is recommended to apply an extra initial dose at 5 weeks of age. Only in cases of outbreak risk during production, booster vaccinations in laying hens and breeders are recommended.
Complete protection in hens is achieved about 15-20 days after administration of the second dose and lasts between 11 and 14 months, according to the type and quality of the vaccine adjuvant. Available vaccines on the market are adjuvanted with aluminium hydroxide gel or double oil emulsions, among others.
Broilers are only exceptionally vaccinated when raised in areas heavily exposed to infection by administering a single dose at the hatchery or later at 15 or 20 days of age.
Various chemotherapeutics and antibiotics have been administered to affected birds via drinking water, often combined with parenteral treatments. When spread of the disease is slow between rows of cages, the birds with symptoms must be injected and re-injected whereas administering medication to the whole flock through water or food. Some examples include the use of enrofloxacin at 10 mg/kg body weight or amoxicillin at 20 mg/kg for at least a week. Combinations of sulfachlorpyridazine-trimethoprim and sulfadimetoxine-trimethoprim via drinking water have also been used, but this administration should be carefully carried out because they may cause kidney damage in the birds.
Often, infectious coryza cases are complicated with Mycoplasma spp.; in such cases an example of a classical treatment that provides good results is the use of streptomycin at 100 mg/kg body weight together with tylosin (30 mg/kg), injecting the combination of both antibiotics subcutaneously. Other parenteral subcutaneous treatments for cases not associated with Mycoplasma spp. are enrofloxacin (20-25 mg/kg) or kanamycin (30 mg/kg) associated with gentamicin (5-8 mg/kg).
Although these treatments have been very successful, there has been an increase in bacterial resistance. For this reason, and considering that antimicrobial susceptibility varies, it is recommended to carry out sensitivity tests in order to select the most appropriate antimicrobial and define strategies for the use of drugs. The indiscriminate and repetitive use of antimicrobials, without testing the degree of sensitivity of the strains present in the farm, promotes bacterial resistance to antimicrobials reducing the effect of the treatment, which also results in unnecessary expenses.
It is important to fumigate (using coarse drops) over the chickens with quaternary ammonium compounds to prevent respiratory transmission, fumigating twice a day. It is important to note that spraying only works as a complementary measure in vaccinated birds but their action is of little help when birds have not been previously immunized.
It should be considered that, after treatment, the infection may be controlled but can never achieve a complete eradication of the disease from the farm, being very important the instauration of biosecurity and disinfection programs. Birds that have become sick, once recovered, act as healthy carriers, so it is highly recommended to treat the animals, perform susceptibility testing for future treatments on the farm and also immunise during rearing all new entering birds into the affected farm.
The authors thank Dr. Fernando Navarro (Veterinary Lazomar, Mar del Plata, Argentina) and Dr. Bernardo Kojic Rousseil (Granja Don Cosme, Buenos Aires, Argentina), for their contributions and technical comments.
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