New Marek's disease cases in Argentina – preliminary studies

Marek´s Disease (MD), a lymph proliferative disorder in domestic poultry that exists in virtually all commercial poultry operations throughout the world, is characterized by mononuclear cellular infiltrates in peripheral nerves and several other organs and tissues (Calnek & Witter, 2000; Schat & Nair, 2008; Witter & Schat, 2003).

MD has been considered the most serious of the many infectious diseases which affect poultry and it is still a problem in Argentina. Studies on Marek's Disease "vaccination failures" have been performed in Argentina since 1988 (Buscaglia et al., 1998; Buscaglia et al., 1999; Buscaglia et al., 2001). Argentina is the second country in the Americas with greater surface area, after Brazil and the eighth largest in the world. It is located in the southern part of South America and occupies a continental surface area of 2,766,890 km² (1,068,302 sq my). It is 5,121 km (3,182 miles) long from North to South, and 1,400 km (approximately 870 miles) from East to West. The poultry industry has approximately a quantity of: 500,000,000 broilers and 33,000,000 laying hens.

Isolated field strains of the MD virus (MDV) in vaccinated batches in the provinces of Buenos Aires, Entre Ríos, Argentina were characterized as highly virulent (VV) strains and very virulent plus (VV +) strains during the decade of the 90´s. The characterization of four Argentine Marek Disease strains as very virulent virus strains was published in 2004, together with the influence of one of these isolates in the synergy between the viruses of the vaccines against Marek´s disease (Buscaglia et al., 2004). Experimental infection with these viruses caused a high incidence of MD in both lines of birds resistant to N-2a and prone to P-2a (Buscaglia et al., 2001;) Buscaglia et al., 2004).

Problems with the emergence of MD in vaccinated batches are still a problem mainly due to: 1) Poor management of vaccines, 2) early and strong exposure to the virus, including the presence of VV+ MDV which can nullify the vaccine immunity. (3) presence of other immunosuppressive agents, such as the infectious anemia virus (IAV) and the reticuloendotheliosis virus (REV), and 4) the addition of antibiotics to vaccines against MD. Different antibiotics (gentamicin A and B and Ceftiofur A and B) were evaluated (Buscaglia personal communication). The commercial HVT vaccine was used. The influence of the addition of an antibiotic on the survival of vaccine viruses against MD was studied. This study was performed to determine the effect of adding the same antibiotic, but from two different laboratories, to commercial MD vaccines and evaluate it in vivo and in vitro. Although it is known that the mix of any antibiotic with MD vaccines is not recommended because it can damage their viability, the addition of antibiotics has become a common practice to prevent early mortality of baby chicks. Results do not show significant differences in HVT titles 30 minutes after been diluted in the presence of Ceftiofur A. or B. Titles after 120 minutes of incubation with product B were slightly lower (Buscaglia & Prada, 2005;) Buscaglia, 2007; (Buscaglia, 2008).

The objective of this study consisted in diagnosing new cases of MD through macroscopic lesions and histopathology and assess the presence of MD in vaccinated populations of laying hens and broilers, the impact on mortality and egg production in the affected farms and its relation with AIPV and VRE continue under study.

History of MD in a company dedicated to egg production.

The company has 4 laying farms and 2 rebreeding farms. Of the latter there is one with 12,000 birds bred on floor near one of the laying hen farms and one more with 40,000 birds reared in cages and more isolated.

The first MD outbreak was recorded in 2005, on the on the bred-on- floor farm. The origin of the outbreak started in birds imported from Brazil from a company that no longer exists. In this re-breeder farm MD outbreaks emerge when the birds are approximately 17 weeks old. During this time several companies provided new birds and the vaccines used were HVT (turkey herpes virus) + Rispens first day (Buscaglia & Viora, 2010). Only in the latest outbreak did birds receive a recombinant vaccine of HVT + Gumboro and mortality started approximately at 6 weeks of age.

Broilers received the HVT vaccine mixed with gentamicin.

Dead or slaughtered birds were autopsied and macroscopic lesions were recorded. Tissues that were questionable were submitted for a Histopathological examination. The tissue was fixed in formaldehyde at 10% and were then included in paraffin and cut. The sections were stained with hematoxiline-eosine (H & E) and were examined under an optical microscope.

The laying hens group that received the recombinant vaccine belonged to a batch of very uneven birds at the time of vaccination. The mortality was 8% the first week, reaching 18% today and showing no signs of stopping or regressing. Comb lesions were observed, the confirmation of which by histopathological studies is in process.

Histopathology showed an abundant cell infiltration in the organs examined. Details of the above mentioned cuts (40 X) showed, in the liver, atrophy of the parenchyma, being reduced to groups of pleomorphic hepatocytes, isolated Among them, with a nucleous-cytoplasma ratio higher than normal. Foci of necrosis and an increase in the stoma due to bleeding, fibrosis and bile pigments and infiltration of small, well-differentiated lymphocytes were also observed. Spleen cuts showed infiltration of the capsule by atypical lymphoid, pleomorphic cells, of rounded or oval nucleus, hyper chromatic and irregular cytoplasm. Cells similar to those described above were found, scattered in the red pulp.

At the pro-ventricle gizzard union the mucosa was manifestly thickened, due to infiltration of atypical lymphoid, pleomorphic cells,, with rounded nucleus and cytoplasm of irregular contour. Nuclei showed a single nucleolus or multiple prominent nucleoli, aberrant mitosis, and variable size. Necrotic, interspersed cells interspersed or foci of necrosis of these cells were also observed. The infiltration affected also the muscular interstitium. Other tissues, including Fabritius bags, nerves and combs have not been examined under the microscope yet.

As set out in this work, MD is still a problem in Argentina. Here we show new diagnosed cases in a vaccinated population of laying hens diagnosed by macroscopic lesions and histopathology.

As in other parts of the world, where the emergence of field strains has exceeded the protection capacity of the trivalent vaccine, the presence of the MDV in strains of increased virulence in Argentina, should be no surprise. Without a doubt, this will have an impact on mortality and egg production in affected farms. The 2 figures (Figure 1 and Figure 2) attached are examples from one of the affected batches.

Figure 1. Mortality curve in a laying-hens farm affected with Marek's disease.

Figure 2. Egg-laying curve in the same farm described in Fig. 1.

Mortality in broilers was 1% daily and at 35 days of age the autopsy also showed aerosaculitis and coryza-type swollen head syndrome. Anyway, MD was diagnosed by histopathology.

MD is still a problem in Argentina. Here we have shown a mortality rate of 15% in a batch of layer hens caused by MD. The outbreak was diagnosed base don macroscopic lesions and confirmed by histopathology, the same case as in the broilers. The decrease in egg production was not significant. The vaccine received by the broilers against MD had been mixed with gentamicin and its effect on the viability of the vaccine is a possible explanation for the outbreak. The evaluation of data belonging to other batches of laying hens and other tissue cuts, is now in process, as well as a possible influence of VAIP and REV.

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