Fall 2018

North Carolina Department of

Agriculture and Consumer Services

Steve Troxler, Commissioner

A Note from Our Director...

We are pleased to

announce that the

Accreditation Committee of

the American Association of

Veterinary Laboratory Diagnosticians (AAVLD) has eval-

uated the report generating from our audit this past July

and voted to grant full accreditation status to our labora-

tory system through December 31, 2023! This is a credit

to our quality assurance program and to all of our staff

that accept and implement this program and work daily to

provide accurate, timely, and reliable results to you, our

valued clients.

Jim Trybus, DVM, DACVP

Foulbrood By: David Ackerman, DVM

Most veterinarians are now familiar with the Veterinary Feed Directive

(VFD) requirements the US FDA put into place in January of 2017. Live-

stock producers and farmers are also aware of the requirements now

needed to obtain and use medically important antimicrobials (MIA) for

their livestock. The Veterinary Feed Directive (VFD) ruling requires veteri-

narians to issue VFDs or prescriptions for MIA within the context of a vet-

erinarian-client-patient-relationship (VCPR) and specifies the key ele-

ments that define a VCPR with livestock producers and farmers.

In the new VFD requirements, honeybees are listed and considered livestock, as beekeepers

produce or collect products intended for human consumptions (honey, pollen, propolis and wax)

from their honey bees. Antibiotics which have been commonly used on honey bees to treated

infectious diseases in the past, without regulation are now listed as MIA, requiring VFD from a

veterinarian. Many veterinarians in the US are not familiar with diseases of the European honey

bee, as they are not routinely or traditionally trained about honeybee health. In North Carolina

(NC) and across the country this has created a potential gap in care for honey bees with bee-

keepers and honey bee inspectors on one side, who are knowledgeable of the management and

care for this unique form of livestock and veterinarians on the other, who are scrambling to obtain

the knowledge to work with them when necessary.

As veterinarians work with beekeepers to diagnose and treat honey bee colonies for disease,

there are many disease conditions which result from management, viral, bacterial, environmental

and parasitic issues, as with other forms of livestock (ruminant, poultry and swine). There are

two bacterial disease which veterinarians need to confidently recognize. More importantly, rec-

ognize the differences to make a correct diagnosis of these diseases. American Foulbrood (AFB)

and European Foulbrood (EFB) are two of the brood diseases, collectively known as “Foulbrood’.

To an untrained eye these diseases may look similar yet require two very different recommenda-

tions and requirements for resolution. Both diseases can cause significant economic losses to

beekeepers if undiagnosed or diagnosed incorrectly.

American Foulbrood

American Foulbrood (ABF) is a highly contagious bacterial disease of the capped brood of the Euro-

pean honey bee (Apis mellifera). ABF is caused by Paenibacillus larvae a Gram-positive spore

forming bacterium. The spores of P. larvae are very hardy and difficult to destroy. When present in

a hive, spores are often found in the wax, honey, pollen, on the bees and the wood frames of the

hive. The spores are spread by the incidentally feeding of contaminated brood food (a mix of pollen

and honey) to the bee larvae by the nurse bees prior to capping for pupation. The spores develop

into the vegetative form in the intestine of the developing larva. The bacteria obtain nutrients and

replicate by digesting the larva. As the bacterium depletes nutritional resources and the conditions

are no longer optimal for the vegetative form, each bacterium develops spores. The dead larvae dry

up into flakes called scales. The scales contain millions of the P. larvae spores. The nurse bees

remove the remains (scales) of the dead larvae, spreading the spores, starting the cycle over. The

colony eventually dies, only be robbed of remaining resources by honey bees from other hives

spreading AFB to other colonies, potentially up to five miles away.

The clinical signs of AFB initially are noted by

a decrease in honey bee population. The

beekeeper notes this as a “weak hive”. At this

point it maybe too late for this colony.

However, a proper diagnosis is important to

prevent spread to other colonies in the bee

yard. Upon approach and opening the hive an

often-distinct odor of rotten meat or dead fish

is noted. Examination of the brood frames

reveal, a spotty brood pattern, many of the

cells which are capped will appear sunken or

have an off centered puncture. The contents

of these abnormal cells are dead immature tan to dark brown, liquefied larvae. Removal of a larva

with a probe from the cell will reveal an amorphous glutinous substance which strings out as a

long-wet thread, > 2 cm. Often called the “Ropey Test”, this observation is considered pathogno-

monic for AFB. Another key observation with AFB, is that the dry scales of the dead larva lie flat

and tightly adherent to lower side of each cell wall. The remains of a pupal tongue are rarely seen

but is indicative of AFB. Confirmation testing is available through the USDA Bee Research Lab in

Beltsville Md. and through the NC Apiary Inspection Services. Samples to be submitted for

laboratory testing need include worker bees, brood and wax comb. These samples need to be

properly sealed when packaged to send to the lab. You can consult with the NC Apiary

Inspection Services or the laboratory of choice on specific requirements. On site diagnostic tests

can also be performed such as the Holst milk test or the Vita AFB antibody test. AFB is difficult to

effectively treat because of the spore forming characteristic of the organism. Treatment and

control therefore are often subject to state laws in the US. Confirmed cases of AFB are to be

reported to the NCDA &CS Apiary Inspection Services in NC. In NC, colonies diagnosed with

AFB are depopulated of the infected colony of bees and brood and equipment is sterilized by

ethylene oxide or destroyed by burning or incineration. Other colonies clear of AFB in the apiary

maybe treated with approved antibiotics dispensed through a VFD or prescription from a licensed

veterinarian. The best practices to control AFB are early detection and confirmation through

frequent hive inspections.

European Foulbrood

When differentiating brood diseases of the honey bee, as there are several of concern, AFB and

European Foulbrood (EFB) can often be confused, as they appear to be very similar. However,

there are differences. European Foulbrood (EFB) is a contagious bacterial infection primarily affect-

ing uncapped brood of the European Honey bee (Apis mellifera). EFB is caused by Melissococcus

plutonius, a Gram-positive anaerobic non-spore forming bacterium. When the uncapped larvae

become infected they can die within a few days. Typically, larvae become infected at 1-2 days of

Photo: beeaware.org au

age by ingesting contaminated brood food from the nurse bees. However, older larva may also be

infected. M. plutonius can only multiple in the midgut of the larval honey bee. The age of the larva

at ingestion of the bacterium and the relative strength, health and hygienic behavior of the colony

will determine the impact of EFB on the larvae and the colony. Most young larvae will die relatively

quickly and will be left uncapped, until removed by the house cleaning bees. Older infected larvae

will die after capping or live and emerge as infected, small or normal size adult bees. Both situa-

tions allow replication and transmission of the bacteria to the next generation of bees. Other

stresses to the colony can impact the effects of EFB on a colony. Unlike AFB, a strong honeybee

colony in the spring may be able to resolve the presence of EFB on its own. Other situations with

EFB require some form of intervention by the beekeeper to recover the colony and prevent spread

within the apiary. Here the key is correct diagnosis of EFB.

Photo Courtesy of Randy Oliver

The clinical signs of EFB initially are noted by decrease

in honeybee population, as with many other brood

conditions. The hive may or may not have a “sour”

odor upon opening, different than AFB. Examination of

the brood frames reveal a spotty brood pattern, with

many cells containing uncapped young dead brood.

Closer evaluation of the cells typically shows yellow to

light brown, often twisted, dead, intact, easily removed

larvae that do not adhere to the cell wall. The larvae

are not glutinous or sticky. The “ropey test” is negative.

Confirmation testing for EFB is also available through the USDA Bee Research Lab in Beltsville

Md. and through the NC Apiary Inspection Services. Samples for submission for laboratory testing

and worker bees and brood. On site diagnostic tests can also be performed such as the Vita EFB

antibody test. Once diagnosed, often resolution of EFB can be managed out of a colony by

strengthening it, by providing additional bees or healthy brood, supplementing (protein) nutrients as

additional feeding and possibly re-queening the colony. Another practice would be to create a

“shook swarm” which is basically removing all the adult bees, including the queen, from the infected

hive, to fresh clean foundation in a clean unaffected hive without any of the brood. The brood re-

maining in the infected hive are destroyed by freezing and the equipment sanitized prior to reuse.

Antibiotic therapy is an option to treat EFB. When necessary, FDA approved antimicrobials can be

used with a VFD or prescription from a licensed the veterinarian for the treatment of EFB in NC. As

with other bee disease, the best practices to control of EFB are early detection and confirmation

through frequent hive inspections by the beekeeper.

Proper diagnoses of EFB and AFB are critical. Unfortunately, over time, some colonies with EFB

have been destroyed needlessly, as they were diagnosed with AFB and worse colonies with AFB

have been diagnosed as EFB or another brood disease and have been managed or treated only to

spread AFB until the colony dies out or been properly diagnosed. Veterinarians have been recently

mantled with a new responsibility involving honey bees, with the new VFD requirements and the

use of MIA. This responsibly has provided an opportunity for veterinarians to work with State bee

inspectors and beekeepers on the health and management of this unique form of livestock.

Veterinarians in NC seeking to embrace this opportunity to work with honey bees can contact their

local NC State bee inspector about honey bees. The inspectors are wealth knowledge and experts

on honey bee health and their diseases. Contact information can be found at:

http://www.ncagr.gov/plantindustry/Plant/apiary/

Additional information for veterinarians seeking education on honey bee health and management

can be found at the links listed below.

https://www.hbvc.org/

http://entomology.ncsu.edu/apiculture

http://www.usfarad.org/honey-bees

http://www.ncbeekeepers.org/

References:

Caron Dewey M. Honey Bee Biology and Beekeeping [Book]. - Kalamazoo : Wiccwas Press, LLC,

2009.

Nicolas Vidal-Naquet DVM, DIE American and European foulbrood diseases [Conference] // Hon-

ey Bee Veterinary Consortium Conference. - Raleigh : [s.n.], 2018.

Ritter Wolfgang Bee Health and Veterinarians [Book]. - Paris : World Organisation for Animal

Health, 2014.

Potomac Horse Fever By: Jennifer Haugland, DVM

In October 2018 , a 9 year old American Quarter horse gelding was diagnosed with Potomac Horse

Fever, also known as Equine Neorickettsiosis, at the Rollins Animal Disease Diagnostic Laborato-

ry. The gelding was from Carteret county and had stood in flooding from Hurricane Florence 4

weeks earlier. In the evening of October 9th, the horse was first noted to be febrile but was other-

wise normal. A veterinarian examined the gelding the next morning and found the horse still febrile

(104.5 F), heart rate of 60 bpm, decreased gut sounds in all 4 quadrants, pale mucous membranes,

and depressed mentation. The gelding was still drinking but not eating that morning. The serum

amyloid A (SSA) was measured as 1008 ug/ml (reported normal interval <5-20 ug/ml). Banamine

was prescribed as needed for the fever. The next day (Oct 11) the manure changed to a cow pie

consistency. On October 12, the gelding had mucous membranes that were red to dark pink, was

severely dehydrated, he appeared disoriented, and the manure had changed to watery. The

gelding died that morning and was presented for necropsy later that day. A necropsy examination

was done the morning of October 13th. Gross exam findings were mild diffuse expansion of the

lungs with pleural ecchymosis. The liver was slightly rounded and the parenchyma was orange.

There was serosal ecchymosis of the small intestine and the mucosa was diffusely dark red to

purple. The dorsal colon mucosa was diffusely dark red with checkerboard like tan lines of fibrin on

the surface (Figure 1). The ventral colon was moderately edematous and there was a diffuse thin

sheet of fibrin on the surface. The contents of the small and large intestine were green and watery.

Microscopically there was a diffuse, mild to moderate, ulcerative, neutrophilic colitis with fibrin and

mild hemorrhage. A mild lymphoplasmacytic portal hepatitis with minimal to mild fibrosis was seen

in the liver. There were no microscopic changes in the small intestine. The lesion in the colon was

most suggestive of a bacterial etiology, although the absence of fibrin thrombi in the lesion put

Salmonellosis lower on the list of differentials.

Figure 1: Dorsal Colon

Small intestine Normal enteric flora No Salmonella isolated No Clostridium perfringens isolated.

Cecum Normal enteric flora No Salmonella isolated Negative for Clostridium difficile toxin A/B No Clostridium perfringens isolated.

Colon Normal enteric flora No Salmonella isolated Negative for Clostridium difficile toxin A/B 4+ Clostridium perfringens isolated, 2 colony types

Kidney No growth after 48 hrs No Salmonella isolated

Liver 1+ E. coli A few colonies of Streptococcus equi subsp. zooepi-demicus No Salmonella isolated

Clostridium perfringens genotyping PCR Both colony types – Type A, non Beta 2 toxigenic and nonenterotoxigenic.

Bacterial cultures did not reveal an etiology. Due to the history of fever and ileus as the initial

complaints and the exposure to floodwater, sections of colon were submitted to Michigan State

University for PCR testing for Neorickettsia risticii, the causative agent of Potomac Horse Fever. The

sample tested positive for N. risticii. This is the first diagnosis of Potomac Horse fever by the NC

Veterinary Diagnostic Laboratory, however, conversations with other veterinarians have revealed

that sporadic cases have been diagnosed elsewhere in the state.

Potomac horse fever is a rickettsial disease that targets the intestinal epithelial cells, mast cells and

macrophages with lesions being confined to the intestinal tract, which are usually underwhelming.

Clinical signs vary considerably and may include fever, depression, ileus, anorexia, laminitis, diar-

rhea, and colic. Colitis does occur in all cases but clinical signs may only be fever and depression

without colic and diarrhea. Diarrhea is only present in less than 60% of cases, is less profuse than

Salmonellosis and may last from 1 day to rarely up to 10 days. Laminitis can vary from mild to se-

vere, sometimes ending in euthanasia. The clinical course of the disease without intervention is 5 to

10 days and case fatality varies from 5 to 30%. Abortions can occur a few months after the mare re-

covers from the illness. Placentitis, fetal colitis, periportal hepatitis, and lymphoid hyperplasia in the

spleen and mesenteric lymph nodes can be present.

To diagnose the disease, PCR testing of feces, colon, whole blood, or fetal tissues is recommended.

Serological analysis is difficult to interpret and thus not usually diagnostic.

Source: Potomac Horse Fever, 2010. Palmer, J.E. New Bolton Center, University of Pennsylvania

https://www.newenglandequine.com/Articles/PotomacHorseFever.pdf)

Streptococcus equi subsp. zooepidemicus By: David Drum, DVM

The body of a 1 month old Friesian colt was presented to our diagnostic laboratory for post mortem

examination. The provided history stated the animal was found cast in the stall and, since being

found cast, was exhibiting increasing severe neurologic signs. The horse had repeated head

trauma due to its neurologic condition and was unresponsive to seizure medications. Uveitis and

corneal ulcer left eye were also present. Euthanasia was elected.

On post mortem examination the horse weighed 77 kg and had a BCS of ~ 2/5. There were multiple

areas of subcutaneous bruising over the cranium. Multiple caseous abscesses present within the

left cerebrum. (Figure 1) The ventral edges of the cranial and caudal lung lobes were red colored

and meaty on palpation. A large caseous abscess was present in the cortex of the caudal pole of

the left kidney. (Figure 2) Subcutaneous and intramuscular bruising was present along the right side

of the neck. The cervical spine was cross sectioned, and no vertebral fractures present. No addi-

tional lesions on examination of the body. Morphologic diagnosis following gross post mortem

examination of the body included: 1) Cerebral abscessation, caseous, extensive 2) Renal abscess,

caseous, 3) Pneumonia, interstitial, regionally extensive. Streptococcus or Rhodococcus would be

among the top differentials for the internal abscesses.

Figure 1: Renal abscess

Figure 2: Cerebral abscess

Strep equi subsp. zooepidemicus was isolated on aerobic bacterial culture of lung tissue and

swabbed abscesses.

Histopathologic examination revealed the following lesions: Brain: Multifocal suppurative menin-

goencephalitis with necrosis, Lung: Marked histiocytic pneumonia with giant cells and abscesses,

Kidney: Suppurative nephritis with necrosis. In the lung tissue, tissue gram stains highlighted

small Gram positive coccobacilli within macrophage cytoplasm, consistent with Rhodococcus

equi, and highlighted Gram positive cocci in pairs, consistent with Streptococcus sp, within the

areas of inflammation, indicating dual infection in the pneumonia.

Veterinary Staff

Director

Dr. Jim Trybus james.trybus@ncagr.gov

Veterinary Diagnosticians

Dr. Jennifer Haugland jennifer.haugland@ncagr.gov

Dr. Stacy Robinson stacy.robinson@ncagr.gov

Dr. Mahogany Wade mahogany.wade@ncagr.gov

Veterinary Pathologists

Dr. Tahseen Abdul-Aziz tahseen.aziz@ncagr.gov

Dr. Steven Rushton steve.rushton@ncagr.gov

Dr. Alison Tucker alison.tucker@ncagr.gov

Dr. Allison Boone allison.boone@ncagr.gov

Director

Dr. Richard Oliver richard.oliver@ncagr.gov

Veterinary Diagnostician

Dr. David Drum david.drum@ncagr.gov

Director

Dr. Heather Wyss heather.wyss@ncagr.gov

Veterinary Diagnostician

Dr. Elise Lavie elise.lavie@ncagr.gov

Director

Dr. David Ackerman david.ackerman@ncagr.gov

Veterinary Diagnostician

Dr. Jessica Kees jessica.kees@ncagr.gov

Rollins Laboratory (919) 733-3986

Arden Laboratory (828) 684-8188

Monroe Laboratory (704) 289-6448

Elkin Laboratory (336) 526-2499