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Human and Animal Health: The Way Ahead
David Franz opened the session by saying that infectious diseases do not respect geographical borders. We also know that they do not always respect boundaries between species. V. M. Katoch mentioned the concept of One Health and that working together across human and animal health professions has considerable value. It is also important for scientists to work across geographic borders, since that is what the pathogens do.
BIOCONTAINMENT SOLUTIONS FOR POULTRY RESEARCH WITH VETERINARY AND ZOONOTIC PATHOGENS
David Swayne focused his remarks on specific aspects of the containment of dangerous pathogens. He and his colleagues1 work under the principle that there are three different components that define containment: (1) facilities or building structure; (2) safety equipment; and, (3) the people and the policies and procedures they follow. Swayne believes that the most important of these are the people. No matter how good the containment facility or the equipment, if people do not follow procedures correctly, there will be problems.
Swayne’s presentation outlined some vital containment situations that he and his colleagues had to face and challenges they had in discussing these issues with other institutions that work with laboratory
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1 Swayne thanked his staff who worked on this project: Andrew Clark who was with U.S. Department of Agriculture/Animal and Plant Health Inspection Service in Egypt at the time; Terry Tumpey at the U.S. Centers for Disease Control and Prevention (CDC). The project was funded by the U.S. Agricultural Research Service and CDC.
animals. Different laboratory animals pose different challenges to the research environment. Birds, which unlike other animals have different eating habits, have different metabolism, and have feathers, not fur, which leads to unique containment issues. First, birds are not mammals with feathers, they are completely different. They have a different kind of metabolism, and consume a different kind of food. Terrestrial birds, such as chickens and turkeys, excrete nitrogenous waste as dry urates. They have a higher body temperature than mammals and, therefore, for example, day-old chicks require a much higher temperature to remain alive. If the temperature is too low, the chicks become stressed, which introduces a variable that is not designed in the experimental protocol. Since these birds have feathers, starting with down and then full pin feathers and finally vein feathers, they can produce abundant feather dander, which can clog filters. For example, equipment with HEPA filters requires several pre-filters, to avoid the destruction of the HEPA filters, and often must be changed mid-experiment.
Generally cages should have negative pressure, HEPA-filtered intake and HEPA-filtered exhaust. The intake air comes from the room most of the time, and the exhaust could go back into the room, but they have ducted the exhaust through the building duct system. The exhaust is HEPA-filtered before it goes in the duct system and the duct system then goes through a double HEPA filter on exhaust.
Other critical aspects that Swayne and his colleagues consider are biosafety and biosecurity. The facility at which he works was built in 1976, when the U.S. Agricultural Research Service actually wrote the manual for facilities and created the category of BSL-3-Ag. According to the select agent program, Swayne’s facility is a BSL-3-Ag facility, although it is not pressure decay-tested because the facility pre-dated the regulations. For over 20 years, the facility had permits at a BSL-3-Ag level. An inspection was conducted in 2008, at which time the lab’s level was reevaluated, and the inspectors determined that the facility would only be a BSL-3 if it did not pass a pressure decay test. As a result, the lab is now designated as a BSL-3 enhanced lab, which means researchers were required to change some of the practices that had been conducted in open rooms. Flexible film isolators, which also have HEPA filters, were installed. They draw air from the room and then they recirculate it back into the room after it goes through the HEPA filter. For example, in the animal rooms, they have necropsy tables encased by flexible film isolators. They may also have freestanding cages of chickens for studies using egg layers; this is designated as a containment area. In this case,
the entire space is considered contaminated, and the researcher wears personal protective equipment when entering the space with the animal.
Swayne then discussed how they use containment facilities to conduct studies with zoonotic H5N1 viruses. Many human cases of H5N1 have been detected, and when epidemiologic studies have been conducted, in approximately 70 percent of the cases the infected person was found to have had exposure to poultry. Generally most of these exposures have been in markets or through household poultry production and slaughter.
Swayne’s group started a project based on research they were conducting in Egypt where most of the H5N1 cases are found in women and children who are the primary caregivers for the household and rooftop poultry. They were trying to understand how the infection occurred, so they partnered with several people to conduct their work. The first experiment was conducted in a room with a concrete floor, concrete walls, and a concrete ceiling. There was directional air flow, HEPA filter intake and exhaust from the room, a shower outside of the room and outside of the building. They had attached an incinerator on their clean-dirty corridor pass-through autoclave. They always wore appropriate PPE because they were in the room with infected birds that they euthanized and processed. These birds were H5N1 inoculated and asymptomatic, which occurs in the first 24 hours after inoculation when they shed a lot of virus.
First they slaughtered the bird on the table. Then they did sampling at three different points in the room at varying distances from the table. Next, they simulated a home Halal slaughter method in five steps: (1) following animal care and use regulations, they tranquilized the birds; (2) once tranquilized, they cut across the carotid jugular; (3) placed the birds into a bucket; even tranquilized, birds still have involuntary muscle contractions for approximately a minute to minute and a half; (4) they hard scalded the carcass at 66 degrees Celsius (150 degrees Fahrenheit) to loosen the feathers; and, (5) then they manually defeathered the birds, eviscerated the carcass, and cleaned up. During the whole process, Swayne’s group ran a negative air ionizing sampler to collect any particles from the air, and later they ran a particle sampler to determine the size of the particles. Figure 3-1 shows the number of virus-infected particles measured at different distances from the table during the first experiment.
SOURCE: David Swayne, presentation at the workshop.
At two different distances from the table, significant numbers of particles were detected during the home slaughter simulation process. They then measured for virus and recovered virus from the air and the farther away they were from the source of the slaughter, the less virus was detected, which would indicate that respiratory droplets are definitely involved in detecting virus. They also put naive chickens in the same air space while they conducted the slaughter process. They found that all of the chickens exposed to airborne virus become became infected and died.
Next, Swayne’s group studied the effects of exposing ferrets, which are the model for human infections, to the avian influenza virus. They conducted the first experiment with the ferrets at 150 centimeters away from the chicken slaughter site. Three of the four ferrets became infected and died of the virus carried by the chickens. Thus, airborne exposure through breathing occurs at the same proximity during the slaughter of infected chickens, and the virus can be transmitted to both chickens and ferrets.
Swayne and his colleagues then investigated how to develop changes in the process to avoid infection. Swayne’s colleague Andrew Clark suggested conducting the slaughter step in a plastic bag. Clark worked for Animal and Plant Health Inspection Service (APHIS) at the time and had traveled to villages in Egypt to observe women slaughter chickens.
He observed that they cut the carotid jugular, and threw the chicken on the dry ground in front of their houses. A large plume of dust could be seen rising around the chicken. Children were often playing nearby and a woman would often stand over the dying chicken.
Clark hypothesized that this was the source of the exposure. Swayne proposed an alternative hypothesis: that the exposure occurred when the women defeathered the birds because there is a great deal of virus in the feather follicles and a vacuum is created when a feather is pulled out of the follicle. When the surface of the skin is broken, the pressure is released and the virus is aerosolized and forms respiratory droplets.
To test these hypotheses in the lab, Swayne and Clark used two groups of chickens, one that had been vaccinated and one that had not. The result was recovery of virus from the oral pharynx and the cloaca of non-vaccinated birds at high titers. The amount of virus recovered from vaccinated chickens was significantly lower. During the experiment, they conducted the standard slaughter on five chickens. They recovered virus in five out of the five non-vaccinated birds from the kill, scalding, and defeathering steps, and then during the evisceration and clean up steps, they recovered virus in four of five non-vaccinated birds.
Next, they switched to conducting the kill step in a bag. The tranquilized chicken was placed in the bag with the head out. The carotid and jugular were cut and the head was placed back in the bag and the bag was placed in a bucket so the chicken flopped within the bag. Using this method, they switched from a negative air ionizing sample to a large impinging sample, and found that there was a reduced number of samples from which virus was recovered following the chicken slaughter and the titers are lower in each of these cases. In the end, they demonstrated proof of principle: if that one step—slaughter—was done in a bag, the amount of virus was reduced greatly. Next, they repeated the experiment using vaccinated birds, which secreted less virus, and they could not recover virus from the air regardless of the process used.
Then they exposed non-vaccinated ferrets to the slaughter of non-vaccinated chickens. Three of three ferrets became infected and died when exposed to the same air space as that of the open slaughter. On the other hand, if the kill step was conducted in a bag, only one of three ferrets became infected and died. With vaccinated chickens, there was no difference in the type of slaughter: no infection in the ferrets was found.
It is clear that vaccination is very important in many parts of the world, but the problem is that vaccination of chickens in the village sector rarely, if ever, exists. Developing countries have abandoned
vaccination of domestic poultry in the village sector and it is largely conducted only in the industrial sector. The other problem is that plastic bags are quite variable in quality.
Following the plastic bag experiments, Swayne’s group considered different slaughter processes, using household items that everyone has available in Egypt, such as a halal pot (a big pot with a lid), and a bucket with a lid. First they used a standard open barrel for the kill step, and then they conducted the kill step in a halal pot, and at the end of a two-minute time span, they lifted the lid off the pot, which they thought might actually create some kind of aerosols and vapors, or aerosols and respiratory droplets.
The other way they conducted the experiment was by placing the chicken in the halal pot during the kill step and then sliding the lid off after some time. Next they used a bucket and placed the bird in it during the kill step and snapped the lid on. The lid had a small hole in the top. Scalding water was poured into the bucket to settle everything out. The experiments with the halal pot and the lidded bucket were conducted using a different table arrangement because over the course of the overall series of experiments, the lab had changed from a BSL-3-Ag to a BSL-3enhanced lab. The experiments were conducted in a flexible film isolator, i.e., a bio-bubble. They also switched to using the Cyclone sampler because they could collect three different particle sizes by dividing aerosols and respiratory droplets in the sampler.
The results using the standard open-barrel method versus the halal pot or bucket and pouring scalding water into the covered receptacle then sliding the lid off demonstrated that they could reduce the size of the larger, respiratory droplets or even reduce the airborne virus below detection levels. This covered-receptacle method seems to have worked. They compared the results when sliding the lid off the pot versus lifting the lid, and there was a bit more virus in the sample from the lifted lid method, but it was not significantly different. Using the modified bucket method, they could not recover virus if they sampled from the air versus sliding the lid off of the bucket at the end of the kill step.
In concluding his presentation, Swayne noted that there are many physical needs for birds that are different from those of mammals, which translates into diverse housing needs. Birds are diverse and housing variations may be needed to accommodate their unique physiological differences.
For infectious disease housing, isolation cabinets are preferred. They aid in maximizing floor space by having more groups in the same space.
If cabinets of the correct sizes are used, researchers and workers are able to reach in and handle the birds easily. This also allows researchers to maximize the number of birds to obtain statistical significance.
In addition, different kinds of enhancements for containment can be used, such as glove port systems. Birds can be transferred through either vaporized acid transfer boxes or through dunk tanks. Swayne and his group prefer to use the large HEPA filter ventilated flexible film enclosures for inoculations, swabbings and necropsies, and other bird procedures such as slaughters.
Discussion
The discussion following Swayne’s presentation focused on specifics of his experiments, religious considerations for the slaughter step, and educating the public.
Jens Kuns opened the discussion by asking three questions of Swayne: How many chickens did Swayne kill at a time for each experiment? Was the anesthesia used a requirement? Did Swayne and his colleagues receive religious input about whether the bucket slaughter would still be considered a proper halal slaughter if it was conducted in that manner, that is, with the hole in the lid? Swayne replied that during the first experiment, ten chickens were killed and then this number was reduced to five; typically five chickens are killed per experiment. He clarified that they tranquilized the birds, and did not use anesthesia. And he confirmed that their collaborators in Egypt took the proposed original project to the Islamic Council in Cairo, from whom they received a letter approving the bird-in-the-bag project and saying this would protect human lives and it did not interfere with halal slaughter.
Swayne continued by saying they have not experimented with the bucket process in the field. Clark returned to Egypt in 2013 and conducted the halal pot slaughters with focus groups of women and it was well received by a large number of participants. Subsequently, they produced posters in Arabic about the process and they are preparing to transfer that material to colleagues in Egypt for distribution. They will try to educate households by educating children who in turn convey this information to their parents. Researchers have found that children have more influence on the adult family members than do case workers distributing posters. Hopefully this will be successful, which may help reduce the number of human H5N1 infections.
HIGH SECURITY ANIMAL DISEASE LABORATORY, BHOPAL: CONTAINMENT OF ZOONOTIC INFECTIONS
Shiv Chandra Dubey began his presentation by noting that the High Security Animal Disease Laboratory (HSADL) in Bhopal, India, was upgraded and designated as the National Institute of High Security Animal Diseases (NIHSAD) in August 2014. Now NIHSAD is an independent institute directly under the jurisdiction of the Indian Council of Agricultural Research (ICAR) and, hence, the institute leaders will be in a position to make decisions as per international requirements.
The lab was designed over a 15-year period with the help of international experts, and with the financial assistance of the Food and Agriculture Organization of the United Nations. After the design and financing phase, it took nearly ten years to fully construct, commission, and validate the facility. Since early 2014, the NIHSAD has been online, and it has delivered the required services, particularly in the wake of avian influenza cases in India.
The institute has a three-floor design. The first floor is dedicated to the air-handling units. The ground floor contains the laboratory as well as the animal wing, and the basement, which is up to five meters down, is dedicated to effluent collection and risk processing. Another special feature is that the majority of the structural engineering components and equipment are of indigenous origin. This has saved a great deal of money, Dubey said, and the overall annual maintenance cost of this lab is less than ten percent of the installation cost. The lab was constructed by the National Dairy Development Board because at that time it was the only agency that had experience with a biocontainment lab, a lab located in Hyderabad which focuses on foot and mouth disease.
The mandate of the HSADL, Dubey noted, includes the building of this facility and developing competence with respect to the handling of exotic animals. HSADL is also responsible for sharing the transferrable technology with partners to help with diagnostics in the wake of or at the emergence of an infectious disease. The HSADL also prepares the biosafety protocols based on indigenous and international requirements; they also educate people about these protocols. Dubey and his colleagues have been associated with training of Indian experts and those from neighboring countries for some time.
The primary biosafety barriers are biosafety cabinets, isolators, personal protective equipment, and personnel training. The secondary barrier includes a double wall structure and a complete cement structure
in the animal area. It also has an air-handling unit for reducing aerosol transmitted pathogens, a rendering plant, and an autoclave. At the time of construction, the facility met all biosafety level 4 (BSL-4) requirements. However, given the advancement of technical requirements for BSL-4 facilitates, the facility is now considered a BSL-3+, and currently there are no plans to upgrade the facility to a BSL-4 level. Since there are annual outbreaks of avian influenza in India, laboratory leaders decided not to renovate the facility at this time because part of the laboratory would have closed and that would have created difficulties in handling the large number of anticipated samples.
After the first outbreak of avian influenza, two facilities were added to HSADL, a specific pathogen-free unit and a transmission electron microscope. During his presentation, Dubey was asked how the response to avian influenza was addressed in such a large country, and he replied that this laboratory is part of the response system that has been developed with the government of India through the Ministry of Agriculture. Fortunately, he said, both the Department of Animal Husbandry, Dairy, and Fisheries, and the Department of Agricultural Research and Education, are in the same ministry that has primary responsibility for international affairs at ICAR. In the event of an outbreak of an exotic disease, the laboratory becomes involved and communicates with World Organisation for Animal Health (OIE) officials. Contingency plans have been developed for specific diseases, particularly avian influenza. Quarantines have been established at the major airports and around-the-clock control rooms can become operational if needed.
HSADL has also been fulfilling its responsibility with respect to diagnostics, training, and active and passive surveillance of samples received at the lab. The animal husbandry departments work in collaboration with the Indian Department of Animal Husbandry, Dairy and Fisheries.
At this point, HSADL has offered these services to the nation for nearly 15 years. Over this time, a few diseases were stopped at the entry point itself, including avian influenza, H7N7, malignant catarrhal fever (MCF), and exotic strains of bovine viral diarrhea (BVD).
HSADL also responded to emergencies following the September 11, 2001 attacks on the United States (9/11) when hundreds of envelopes suspected of containing anthrax spores were sent to HSADL for diagnosis. Fortunately, no anthrax was found. The lab has also been able to confirm the existence of certain diseases not native to India, based on clinical confirmation. The lab was also able to confirm the existence of
avian influenza, both low-pathogenic and highly pathogenic; BVD, MCF, and Bovine immunodeficiency virus infection; swine influenza in 2010; border disease virus in 2011; and West Nile fever and Crimean-Congo haemorrhagic fever (CCHF) in 2011. Routinely, screening for many other diseases occurs at this lab.
Since 2006, Dubey stated, nearly 0.8 million avian influenza samples have been handled at HSADL and have been reported to the OIE. Over the years, the virus has been found in nearly 13 of the 29 states of India. The work carried out at HSADL revealed that until 2007, the declared virus clade of the H5N1 was 2.2; then, after an outbreak in Myanmar, it changed to 2.3, and based on a 2010 isolate from Nepal, they declared the virus clade to be 2.3.2. The clade of the virus detected so far since 2011, in Bhutan, India, and Nepal has come to 2.3.2.18 so this has been the overall molecular composition of isolates handled at HSADL, which has been reported internationally.
In India, crows have been central to the epidemiology of avian influenza. Samples from crows have been received at the lab and the virus was isolated and confirmed. However, there have been no zoonotic implications thus far in India, whereas there have been zoonotic cases in Bangladesh, Myanmar, Pakistan, and other neighboring countries.
The emergence of amantadine resistant influenza A viruses has also been detected by scientists at the lab. Before 2010, CCHF was considered nonexistent in India, but the lab positively identified it in tick samples from Gujarat after a CCHF outbreak in humans between 2010 and 2012. In addition, antibodies of West Nile fever have been detected in ducks in northeast India, and MCF has also been detected in a few places.
After ten years of working at HSADL, Dubey believed it important to work with experts to prioritize the establishment of laboratory guidelines. He led a brainstorming session on guidelines and experts recommended that the lab should have preparedness and surveillance functions for certain diseases and diagnostic preparedness for a number of other diseases, which have not yet been tested or which have been tested serologically.
Dubey and his colleagues have prepared a proposal with technical details on upgrading the lab, and submitted it to the government of India. The plan has been approved and the funds have been allocated, and upon the completion of this plan, the HSADL will have two integrated BSL-4 facilities and the older lab will continue to operate as a BSL-3 facility.
SOURCE: Shiv Chandra Dubey, presentation at the workshop.
After a great deal of discussion and after political and legal matters have been addressed, the new, integrated lab in Bhopal can go forward (see Figure 3-2).
One side of the facility will contain the laboratory, and the other side will contain the animal wing. There is a series of showers and sprinklers next to the lab. The lab has a controlled entry and a clean corridor for entry to the lab from the showers. There are two changing rooms, an entry changing room as well another changing room inside the lab with a shower in between.
The HSADL was a unique effort by the government of India, particularly ICAR, to build such a facility prior to the existence of the current understanding of biosafety. It was difficult to convince politicians and administrators, which is why it took 15 years for the development of the plans and 10 years for the construction for the facility.
Discussion
The discussion following Dubey’s presentation focused on international collaboration and community acceptance of the lab.
International Collaboration
Indira Nath noted that when she saw the data presented by the WHO representative at the workshop reflecting the paucity of BSL-3 and BSL-4 labs in the South Asian region, she wondered whether other countries have access to Dubey’s lab, and if there are any problems. Dubey replied that they do receive samples from the other countries. After receiving influenza disease status from the OIE, neighboring countries can have access to HSADL. Also international experts are trained at HSADL, OIE, and by other organizations. This is a regular practice and the epidemiological information received through HSADL processing is provided to neighboring countries, and results are provided as quickly as possible. With respect to Bhutan, when a sample is received at 11:00 a.m., the results are returned via email by 4:30 or 5:00 pm that same day. During peak days of the Bhutan outbreak, scientists and the team at HSADL worked for 16-18 hours a day, and the overall handling of samples sent was very good.
Nath further asked about the bureaucracy involved when international scientists want to access Dubey’s lab. If a scientist in Nepal would like to have a sample tested, does he/she have to contact the Ministry of External Affairs or the Ministry of Health? Dubey replied that the guidelines are similar to those of the government of India and the guidelines for transport of samples follow OIE procedures. The NIHSAD website clearly details ICAR guidance regarding the type and size of individual disease samples that can be sent, how they should be packed, and how they should be transported.
Gaining Acceptance from the Local Community
Franz asked about community concerns regarding the building of the lab. Dubey replied that one of the politicians from Uttar Pradesh who was a minister in the government of India and former chief minister of Uttar Pradesh was involved, and he was able to address community concerns. Also, whenever there were difficulties from the administration or from various other agencies, he was always involved and his involvement directly or indirectly was able to convince the other politicians and administrators to solve the problems. Dubey noted that
