Effects of the Soil-Derived Microorganism BX-1 on Chicken Newcastle Disease

In recent years, effective microorganisms (EMs) have been administered to humans and domestic animals, and their usefulness has been recognized for promoting health and enhancing immunity. For example, the preventative effects against flu are enhanced by ingestion of Lactobacillus by humans, and symptom relief of atopic dermatitis has been reported, with EMs actually used in commercial products. In addition, EM preparations are being used in livestock to prevent infections (e.g. Salmonella and Escherichia coli infection). In poultry, avian influenza and Newcastle disease are terrible and fatal infectious diseases that cause significant economic damage. Furthermore, countries designated as contaminated with these pathogens can experience major trade problems. Given the above, how to protect livestock from infections safely and at low cost without using disinfectants, antibiotics and vaccines is a major issue. In the present study, we examined whether or not Newcastle disease could be suppressed by feeding chickens BX-1 as an EM feed. A field strain of Newcastle virus was cloned from cloaca swabs of large numbers of dying chickens in a poultry farm in Indonesia by polymerase chain reaction (PCR) and hemaggregation assays. Chicken kidney cells and embryonated eggs were highly sensitive to this virus, and high titers of virus were able to be collected. The experimental viral inoculated to chickens showed a high mortality rate, with high pathogenicity in birds. Conventional chickens were also raised on a diet supplemented with BX-1 and directly infected with the Newcastle virus. The mortality was decreased in these infected birds. Even the low dose of BX-1 had an inhibitory effect on the lethality of the infection. These results suggest that BX-1 intake through an EM diet is effective in controlling Newcastle disease.


Introduction
Newcastle disease (ND) is a viral infection to which various species of bird, including poultry, are susceptible [1]. ND outbreaks occur in many countries, including Japan, and the disease is highly contagious exerting a significant economic impact. In Japan, ND is designated an infectious diseases under the livestock infectious disease prevention law for chickens, ducks, quails and turkeys [2]. Other infectious diseases mentioned in this law for poultry include highly pathogenic avian influenza, poultry cholera and salmonella infections. others. NDV infection occurs through feed, water, equipment and clothing contaminated with feces of affected birds [3]. Symptoms vary greatly depending on the NDV strain, host species, health status and age, among other factors, but common symptoms include those of the respiratory system (dyspnea, cough) and nervous system (depression, loss of appetite, wing weakness, paralysis), swelling of the eyes and neck, diarrhea, abnormal egg shells and decreased egg production [4].
NDV strains are divided into highly toxic, addictive and attenuated strains.
The highly toxic type is further divided into an Asian type and an American toxic type [1]. Severely virulent strains cause serious respiratory and neurological symptoms, are highly infectious and have a mortality rate of over 80%. Addictive strains cause coughing and egg-laying abnormalities, resulting in a mortality rate of about 10%. The mortality of attenuated strains is negligible. Since the countries that use ND vaccines are designated as contaminated countries, their export of poultry products, such as chicken and eggs, to clean countries, such as the USA and Canada, is not permitted. If a novel ND prevention method could be developed instead of a vaccine, the livestock industry would benefit from a trade perspective.
As a probiotic, the mixed microorganism agent BX-1, which is mainly composed of Lactobacillus and yeasts removed from soil, has been commercialized (Kawashima Co., Ltd.) [14]. Since BX-1 is a mixture of soil microorganisms, it does not seem to exert the same effect as the natural intestinal microflora; nevertheless, it can still be expected to be useful as a member of the bacterial microflora with many physiological activities that can benefit the living body. BX-1 is a group of aerobic bacteria, and since it is in a dry powder form, it is easy to handle and inexpensive. BX-1 has been suggested not only to suppress the colonization of intestinal pathogens, such as Salmonella and Escherichia coli, but also to enhance the vitality of the living body, particularly the immunity.
We herein report the results of our infection experiment using a highly pathogenic field strain of NDV obtained from a poultry farm in Indonesia.

Materials and Methods
Newcastle disease virus ( Effects of BX-1 intake in chickens infected with NDV farm strain BX-1 powder mixed at 0.02%, 0.2% and 2% (w/w) in commercial chicken formula feed was fed to 10-day-old white leghorn chickens. Over 10 individuals were used for each BX-1 formulation dose. As control animals, other chickens were fed the same commercial diet without BX-1 powder (0%). At one week of BX-1 feeding, 8 HA of the NDV farm strain was been inoculated intranasally into each chicken. These animals were raised under free water and feeding conditions in a BSL-2 facility, and the mortality rate was confirmed by the 10th day.

Results
Organisms in BX-1 The species of organisms in BX-1 were confirmed by a third-party laboratory in Japan and listed in Table 1 (Figure 1).
In addition, the solution from farm could aggregate the chicken erythrocytes which were inhibited by the addition of anti-NDV antibody ( Figure 2). Over 80% of chickens died following intra-nasal inoculation of the CAF. Histopathologically, ND-specific findings, such as severe acute pneumonia and tracheitis, myocarditis and acute enteritis, were observed ( Figure 3). Accordingly, the pathogens obtained from the Indonesian poultry farm were confirmed to be a highly pathogenic strain of NDV.    In the heart, marked inflammation is observed with mucosal epithelial necrosis and heterophil infiltration (B). In the heart, extensive myocardial necrosis and interstitial inflammatory cell infiltration (such as heterophils) are observed (C). Bar, 100 μm.
Effects of BX-1 intake on ND in chickens Viral solution (8 HA) of NDV farm strain was inoculated into the nasal cavity of 10-day-old chickens, and the number of deaths was counted for the next 10 days, with or without BX-1. As a result, the mortality rate of ND-infected chickens drastically changed in the flock fed the BX-1 diet for one week before virus inoculation (Figure 4). Even the lowest dose of BX-1 at 0.02% exerted an inhibitory effect on mortality. Furthermore, higher doses of BX-1 tended to slow the deaths of birds. Given the lack of any marked differences in the mortality among BX-1 dosages at day 10, implementation of BX-1 for practical use at a poultry farm was considered feasible even at a dose of 0.02%, with consequent cost reduction expected. Since 0.2% -0.5% BX-1 is used as the commercial products to prevent Salmonella colonization in chicken intestinal tracts, this commercial product is effective on the prevention for chickens by both bacterial and viral infections [14].

Discussion
One particularly interesting and potentially important finding from this study was the observation that BX-1, a soil microbial EM agent, was able to control not only bacterial infections, but also viral infections: the intake of soil bacterial preparation BX-1 reduced the mortality rate of chickens infected with highly pathogenic NDV. Soil bacterial EM preparations can be mass-produced, are excellent for storage and transportation and can be supplied at low cost [13]. Therefore, EM is considered useful as a feed additive for livestock.
Since ND is a viral disease and thus operates differently from bacterial dis-  [8]. More detailed cellular and molecular analyses will be required in order to verify the mechanism underlying the ND-suppressive effects of the soil bacterial group BX-1.
In nature, birds commonly ingest soil and thereby consume various microorganisms from the environment. In poultry, however, breeding conditions lacking any windows or having only half-windows are becoming increasingly common, and therefore chickens often no longer consume soil. The construction of a ground-based poultry farming system might be useful as a countermeasure against infectious diseases.