Biosafety Measures Against Agriculture Pathogens of Pakistan

Biosafety measures against all types of pathogens like bacterial, viral, fungal,
nematodes etc

In comparison to public health standards for humans, there are different guidelines for

management and assessment of risk in case of agriculture. For risk assessment and

management in agriculture, the possible impact of morbidity of plants and animals on

economics is taken into account as well as other factors such as mortality and trade

implications of disease. Greater importance is given to the agent to keep it from escaping into

the open environment where it can cause further problems. Unique to agriculture: BSL-3-Ag,

the different parameters of facility and work practices will be explained in this article along

with agriculture pathogens.

BSL-3-Ag for Work with Loose-housed Animals

When a research involves certain risky livestock pathogens, biocontainment of a special kind

is required whereas sometimes the room where the research is being conducted provides the

primary containment. A special facility operated at animal containment level has been

developed by USDA, and is known as BSL-3-Ag. Environment is protected using all the

facilities from BSL-4 level enhancement. It can be a separated block or simply a zone that

has been isolated from rest of its surroundings which can operate at BSL-3 level, safely.

“Box within a box” principle is applied to it and access to it strictly controlled. ABSL-3

along with some enhancements made to BSL-4 for those BLS-3-Ag facilities that are unable

to immediately house animals in primary containments:

1. Personnel change rooms along with shower rooms: Separate clothing is a must for

workers that are working with these animals. Certain ventilated vestibules plus a

change room right outside the containment is required. A shower room should be

installed at the point where containment and non-containment areas meet. A set of

entire animal facility clothing is provided that is always kept inside the dirty change

room of the containment. After leaving the area of potential high risk, workers change

their clothing in dirty rooms and take showers and then move to the clean rooms to

put on lab clothing. Before leaving the facility, another shower and set of clean

clothes is mandatory. The dirty clothes are autoclaved.

2. Access doors and emergency exists: Access doors are lockable and are

automatically closed. Several emergency exits are provided. However, no entry is

possible from outside against unauthorized usage.

3. Entry of equipment, materials and supplies: At entry points for supplies and

equipments, fumigation chambers that are air locked followed by double door

autoclaves that interlocked are installed.

4. Double-door autoclaves for waste decontamination: Situated within the exterior

walls of the containment, these are made with bioseals and have double doors that are

interlocked and the outer door can only be opened after completion f the process of

sterilization. Materials that are disposable are autoclaved and then incinerated.

5. Ventilation systems: Each BSL-3-Ag facility has its own air supply and exhaust

system with a directional flow of the air. Direction of the air flow is such that it

moves from lowest potential hazard area to highest potential hazard area. At insides

and outsides of the containment a visible pressure differential display is present.

6. HEPA filtration: All the air coming to or from the containment area is HEPA

filtered. These filters are located as close as possible to the containment space. HEPA

filters for high level bioconatiments are installed in series as well as in parallel on the

air exhaust sides. For areas of high risk, pre-filters with efficiency of maximum 90%

are installed for supply and exhaust air systems. The air provided outside the

containment zones should be 100% conditioned.

7. Liquid waste sterilization system: Liquid releases are to be decontaminated before

their disposal into the sewers. Heat decontamination system is commonly used which

eliminates any biohazardous material when subjected to controlled high temperature

and a certain pressure for certain time. Liquids from shower rooms, sinks of labs etc

can be decontaminated by adding chemicals as well.

8. Airtight surfaces: this includes airtight sealing of all the interior surfaces as well as

penetrations of all the rooms\spaces within the containment and the boundaries of

containment itself. This allows no contamination happening between the separate

rooms within the containment space.

9. Airtight ductwork: all the ductworks that lead to or from the containment should be

airtight and biosealed.

10. HEPA filters: these should be present at all the possible atmospheric vents that serve

plumbing traps. Liquid disinfectant is filled inside all these traps.

11. Hinges/knobs: hinges and knobs of all access doors are to be sealed airtight.

12. Air inflated gaskets: These are must haves at the all the airlock doors. Further, some

check valves and HEPA filters are installed along with these.
 13. Pathological incinerators: These must be provided for the safely disposing infected

animal remains.

14. Restraining devices: these are installed in animal rooms that are large in size.

15. Necropsy rooms: These rooms are spacious enough accommodate farm animals of

larger sizes and all required equipments are installed within them.

16. 16. Biosafety Cabinets: if installed any, their location must be considered carefully

as air circulation can affect them and BSCs class two needs HEPA filters and a good

exhaust system.

Summaries of Selected Agriculture Agents in Pakistan

Viral Agents

African Horse Sickness Virus (AHSV)

Agent: genus Orbivirus in the family Reoviridae. It has 9 serotypes.

Hosts: Chicken egg, cell culture, sulking mice, horses, donkeys, some mules and sometimes

dogs.

Regions of occurrence: Central Africa, South Africa, North Africa, Middle East, India,

Pakistan and Afghanistan.

Transmission: Transmitted by Culicoides species and other insects.

Occupational Infections: 4 lab workers infected and showed symptoms such as encephalitis

and uveochorioretinitis.
Laboratory Safety and Containment Recommendations: Found in spleen, lymph nodes

and lungs. It can only be worked on with enhancements in both in vitro in a BSL-3 laboratory

and in vivo in a USDA-approved ABSL-3 animal facility.

Special Issues: Importation, possession, or use of this agent is prohibited by law. A special

permit is required for it.

Contagious Caprine Pleuropneumonia Agent (CCPP)

Agent: M. mycoides capri (type strain PG-3).

It causes an acute, highly contagious disease in goats. M.

mycoides mycoides was isolated from goats that suffered from

pneumonia.

Regions of occurrence: Africa, Eastern Europe, the Middle

East, Far East and Pakistan.

Transmission: Inhalation of infective aerosols

Laboratory Safety and Containment Recommendations: Not a

pathogen of humans. However the agent can escape into other Figure 1: Infected horse.

goat populations and infect them. Workers are only allowed to be

worked on in vitro in a BSL-3 laboratory and in vivo in a USDA-approved BSL-3-Ag facility

for loosely housed animals.

Special Issues: importation, possession, or use of this agent is prohibited.

Use requires a permit.

Figure 2: Lungs of a goat infected with CCPP

Peste Des Petits Ruminants Virus (PPRV)

Agent: Peste Des Petits Ruminants Virus

Hosts: Goats, sheep, water buffalo, camel.

Regions of occurrence: West Africa, Arabian Figure 3: Dried areas around eyes and mouth due to the
infection.
Peninsula, the Middle East, and the Indian

Subcontinent. It affects gastrointestinal and respiratory

systems resulting in 90% morbidity and 80-100% death rates.

Transmission: close contact between animals.

Laboratory Safety and Containment Recommendations: no infection in humans is

caused by this agent. The only risk is this virus escaping into outside populations. BSL-3

laboratory for in vitro and BSL-3-Ag facility for in vivo is need

Special Issues: A special permit is required for its sample or when working with this virus in

labs.

Sheep and Goat Pox Virus (SGPV)

Agent: capripoxvirus

Hosts: Sheep and goat

Regions of occurrence: Africa, the Middle East and most

parts of Asia including the Indian Subcontinent.

Transmission: Direct contact and inhalation of aerosols.

Figure 4: pox lesions on a goat skin.

Laboratory Safety and Containment Recommendations: Not a pathogen of humans. Risk
is same as before that is agent escaping the lab and spreading in local cattle
populations. It can only be worked on in BSL-3 laboratory or ABSL-3 animal facility.

Special Issues: Needs special permits otherwise its use, transportation etc is prohibited.

Bacterial Agents

Bacillus anthracis

Agent: Bacillus anthracis

Disease: Anthrax which is an acute disease caused by this agent in mammals

Hosts: Most mammals (especially herbivores, also effect carnivores)

Regions of occurrence: Asia, Africa, US and other regions of world too.

Transmission: direct contact, Inhalation or ingestion of infectious spores.

Occupational Infections: numerous cases from labs have been reported. Infections mostly
occur due to careless contact with the infected organism.

Laboratory Safety and Containment Recommendations: Can be present in blood, animal

dung and other body fluids. BSL-2 and ABSL-2 practices are required whenever working
with this agent. BSL-3 practices are required when high concentrations of the infectious
agent are being dealt with. All instruments, equipments involved must be autoclaved.

Special Issues: A licensed vaccine is

available. It is a select agent and therefore
its possession or transfer within a country
requires registration with the respective
authorities. A permit is needed for
Figure 5: Bacillus anthracis.
transportation and all the rules and
regulations required for international
transfers are applied.
Burkholderia mallei

Agent: Burkholderia mallei, Gram –ve rods

Disease: Glanders

Hosts: horses, goats, donkeys, Humans

Regions of occurrence: Asia and the eastern Mediterranean

Transmission: inhalation and mucocutaneous exposure, Zoonotic transmission to humans.

Occupational Infections: those working with equine sp. Or are directly dealing with the B.

mallei cultures usually contracts the diseases.

Laboratory Safety and Containment Recommendations: it is a high risk pathogen in labs.

BSL-2 practices for primary isolations of fluids from infected persons. BSL-3 lab required

for furthering working with those acquired fluids. ABSL-3 practices are must when working

in animal facilities.

Special Issues: requires a special permit and registration with respective authorities of the

country for possession and transfer of this agent.

Clostridium tetani

Agent: Clostridium tetani, anaerobic gram-positive rod. It produces a neurotoxin which is

known as tetanospasmin.
Figure 6: Clostridium tetani

Disease: Tetanus

Transmission: wound getting contaminated by soil. As the bacteria are soil dwelling, it

enters through broken skin.

Occupational Infections: this is not a high risked pathogen so not many cases of such

infections have been reported.

Laboratory Safety and Containment Recommendations: majorly found in soil. An

accidental inoculation can happen to a lab worker and that is the primary hazard. BSL-2 and

ABSL-2 practices in case of animals are to be observed.

Special Issues: vaccines are available and given to lab workers that deal with the pathogen.

Transportation of the pathogen requires special permits from respective authorities.

Fungal Agents

Histoplasma capsulatum
Agent: Histoplasma capsulatum

Disease: Histoplasmosis

Host: Humans

Regions of occurrence: worldwide

Transmission: Inhalation of infectious particles, inoculation by accident during lab work.

Occupational Infections: It has been transmitted to the lab workers several times during

working with its culture or when performing autopsy of an effected person.

Laboratory Safety and Containment Recommendations: BSL-2 and ABSL-2 practices

Culture samples should be studies under Class II BSC.

BSL-3 practices are required when working with the mold form of the sporulating agent.

Special Issues: Domestic transportation and importation requires special permits.

Sporothrix schenckii

Agent: Sporothrix schenckii, dimorphic fungus.

Disease: sporotrichosis

Host: Humans
Regions of occurrence: worldwide

Figure 7: Skin infection by Sporothrix schenckii

Transmission: inhalation, common exposure to a point source.

Occupational Infections: accidental inoculation of the infectious material has been reported

as the main cause of infections. Skin and eye infections are quite common.

Laboratory Safety and Containment Recommendations: commonly reported are eye and

skin infections however it can cause severe infections in immunocompromised

people/workers.

BSL-2 and ABSL-2 practices with class two BSC are the basic requirements.

Special Issues: Domestic transportation and importation requires special permits.

Intestinal Protozoal Parasites

Entamoeba histolytica, Cryptosporidium, Giardia & Isospora: cause most occupational

infections and is a serious threat to lab workers.

Enterocytozoon bieneusi & Septata intestinalis : both comes from the family microsporidia
and are major intestinal pathogens along with the above mentioned three.

Isospora belli, Cryptosporidium parvum & C. hominis: cause isosporiasis &

cryptosporidiosis respectively.

Entamoeba histolytica: cause amebiasis and can affect intestine as well as extraintestinal
regions.

Giardia intestinalis: disease caused by this is known as giardiasis.

Occupational Infections: many infections with the above mentioned species has been
reported in lab personnel. The agent mimics the natural routes of infection and that is why
the laboratory acquired infections also look like they were naturally acquired.
The common clinical symptoms are that of gastroenteritis which includes cramping, appetite
loss, diarrhea and nausea etc.

A person infected with E. histolytica may have blood in their stools.

These infections in lab can be acquired through animal sources if the lab personnel are
working with any. This usually happens through contact with the feces of infected animals.

Eggs of Cryptosporidium require careful handling. Most lab related infection is due to this
agent.

Further, airborne transmission of its small eggs can also occur. If lab protocols are followed
strictly, the chances of getting infected with this agent reduce greatly.

Transmission: These infections can occur worldwide. Main route through which this agent
is transmitted is ingestion of its oocysts. It multiplies inside its host therefore even a small
inoculation can cause infection easily.

C. hominis: main host is humans

C. parvum: multiple hosts including cattle, humans etc

E. histolytica: major host is humans

I. belli: humans are the main hosts for it as well.

Giardia spp: take both animals and humans as their hosts. Human-human transmission has
been seen with this agent.

Drinking water contaminated by these agents is another common route for infection
transmission.

Laboratory Safety and Containment Recommendations: people with suppressed or weak

immunity are at more risk. Accidental ingestion of the parasite is the main reason for lab
acquired infections. If lab workers are only dealing with the killed parasite then the risk of
infection is near to none.
For Sarcocystis accidental infections with it has not been reported.

Nevertheless, there is always some risk associated with it. Therefore, certain precautions
should be taken even when the apparent risk is very low.

BSL-2 and ABSL-2 practices must be adhered to strictly. Required facilities and containment
equipment are strongly advised when involving in activities that require some kind of contact
with the infective agents.

Oocysts of Cryptosporidium are usually present in stools and are very infectious.

E. histolytica & G. intestinalis: Iodine disinfectants can effectively kill these agents. High
conc., of chlorine can work well too.

To clean a spill containing oocysts Cryptosporidium

 A detergent made especially for labs should be used.

 All the surfaces where the splashes from spill might have settled should be cleaned
thoroughly.
 Hydrogen oxide can be used to clean organic substance.
 It can also be used to disinfect other surfaces.
 Or one can just flood the contaminated surfaced with hydrogen peroxide and then let
it sit for a while before cleaning it.
 Contaminated items can be heated and washed with a strong detergent or they can be
autoclaved it possible.

Special Issues: infections with parasitic protozoa can be treated with medications and
effective treatments are available.

For Cryptosporidium, a treatment is available but it’s not very efficient and further treatments
for it is under development.

Domestic transportation of parasitic protozoa may require permit. Importations and exporting
it to other countries require permissions from respective authorities and all the international
rules and regulations regarding transportation of such agents must be followed religiously.
Trematode Parasites

Schistosoma spp: greatest hazard as it causes the most occupational infections.

Schistosomiasis is caused by these species. This disease is also known as Manson’s blood
fluke.

Fasciola: not as dangerous as former species but still of concern. These cause fascioliasis
which is otherwise known as sheep liver fluke.

Hosts: humans, sheep, dogs, snails etc

Occupational Infections: F. hepatica, S. mansoni & Schistosoma spp all can cause lab
acquired infections.

F. hepatica: usually infections that do not show any visible symptoms.

Transmission: ingestion such as intake of uncooked vegetables which carried the agent's
encysts. Their cercariae easily penetrate the skin as well.

Regions of occurrence: almost worldwide but Schistosoma mansoni is wide spread in South
America and Africa.

Laboratory Safety and Containment Recommendations: their infective stages are present
on leafy plants. When working with such plants, lab workers should take extra precautions as
some species can penetrate through skin easily. Direct contact with the infected hosts should
be avoided.

Schistosomiasis has been the main case reported by almost all the labs that got this infection.
Other infections are not very common in lab workers. But accidental infections with any
species can occur nonetheless.

BSL-2 and ABSL-2 practices must be adhered to strictly. Required facilities and containment
equipment are strongly advised when involving in activities that require some kind of contact
with the infective agents.
Special Issues: for trematode caused diseases, effective medical treatment is available.
Transportation of any kind of this agent may require a special permit from the respective
authorities of the country.

Nematode Parasites

Baylisascaris and Ascaris: two main nematode parasites that cause most occupational
infections in humans.

Hookworms: effect humans as well as animals

Strongyloides: humans & animals, cause strongyloidiasis, commonly known as threadworm.

These live in small intestine in their adult form.

Enterobius; cause enterobiasis, commonly known as pinworm of humans, stays in cecum and
appendix.
Brugia & Wuchereria: human filariae.

Ancylostoma braziliense & A. caninu: main cause of infections in dogs and cats.

Occupational Infections: A. lumbricoides, E. vermicularis, Ancylostoma spp and

Strongyloides spp. infections usually happen in laboratories. Strongyloides and hookworm
infections are usually acquired from effected animals during lab work.

These infections are usually asymptomatic.

Figure 8: Hookworm

Transmission: A world wide endemic is caused by Ancylostoma. It can penetrate the skin
and cause infections in humans. Infection in dogs and cats is endemic worldwide. Human
infection occurs through penetration of the skin. When dog\cat hookworms penetrate the
skin, creeping eruption happens. It can also infect if it gets ingested. However, larvae are
unable to reach intestines.

Another endemic in tropical areas of the globe is the infection caused by Ascaris
lumbricoides. Accidental Ingestion of the eggs of this nematodes results in infection. Those
Figure 9: Ancylostoma braziliense
eggs that have not been embryonated yet
would pass in stools and further needs 2-3
weeks to turn into their infectious form.

Worldwide infection occurs due to

Enterobius vermicularis. School age kids
are more susceptible to it that adult humans.
The infections usually happen after
ingestion of the worm eggs. These eggs,
however, requires hours to become active
 infectious agents. Infection is not very long lasting but reinfection can keep the cycle going
on for long.

Strongyloides infects dogs and cats and usually happens as endemic worldwide. Humans can
get the infection too if the infected particles come in contact with broken skin. This results in
the formation of creeping eruptions on the skin.

Laboratory Safety and Containment Recommendations: ingestion of the eggs or larvae of

nematodes or their entrance into the body through broken skin is main hazard. Most
nematodes’ eggs are not infective at early stages. They require some development that might
take several days to several weeks depending on the outside conditions and the nematode
species. Hypersensitivity can develop in laboratory worker if
Figure 10: Creeping eruption or cutaneous larva
migrans. frequently exposed to the ascarids’ infective aerosolized
agents.

Ascarids eggs should be handled carefully and it should be made ensure that everything
including all the equipments that came into
contact with these is later decontaminated
completely. These eggs are somewhat
sticky. Stool samples that Formalin-fixed
are not be treated risk free because these
eggs remain infective even in the formalin
and they even develop in it.

Infective Eggs of Toxocara and

Baylisascaris, can cause visceral migration
of larvae which can further attack central nervous system and eyes

Infective eggs of Strongyloides stercoralis can badly affect people who have weak immunity
or suppressed immunity. It can lead to life threatening conditions as a severe form of
infection can occur which is called hyperinfection.
The surfaces or instruments that have been contaminated accidently can be sterilized using
the Lugol’s iodine. It is very effective in killing the larvae that is infectious The larvae of
Trichinella’s larvae if ingested by accident can cause serious infection.

Arthropods that are infected by these worms are also a potential hazard to lab workers.

BSL-2 and ABSL-2 practices must be adhered to strictly. Required facilities and containment
equipment are strongly advised when involving in activities that require some kind of contact
with the infective agents. Infectivity of nematodes is different at different stages of life so
one needs to know the each stage in order to play safe. Inhalation of aerosols that are actually
the ascarids antigens is a hazard to health.

Special Issues: very effective treatment for almost all nematode infections is available.
Transportation within the country or outside the country, Importation etc may require a
permit from respective authorities in the country.

References:

 Chosewood, Lewis & Wilson, Deborah. (2009). Biosafety in Microbiological and

Biomedical Laboratories, 5th Edition. US Department of Health and Human Services.
 List of Animal Diseases in Pakistan. 19th November, 2019. Invasive Species
Compendium. CABI.
https://www.cabi.org/isc/datasheet/108537#tolistOfAnimalDiseases
 Mattos, G. T., Santos, P. C., Telmo, P. L., Berne, M. E., & Scaini, C. J. (2016).
Human Toxocariasis: Prevalence and Factors Associated with Biosafety in Research
Laboratories. The American journal of tropical medicine and hygiene, 95(6), 1428–
1431. https://doi.org/10.4269/ajtmh.16-0196
 Parida, S., Muniraju, M., Mahapatra, M., Muthuchelvan, D., Buczkowski, H., &
Banyard, A. C. (2015). Peste des petits ruminants. Veterinary microbiology, 181(1-2),
90–106. https://doi.org/10.1016/j.vetmic.2015.08.009