Updates from Ohio State and Camelid Parasite Diagnosis, Control, and Prevention

Rebecca Pentecost, DVM

Hospital for Farm Animals

Veterinary Clinical Sciences

Objectives of Lecture –

• Updates from Ohio State University College of Veterinary Medicine

• Discuss one of the most important problems facing the Camelid producer

• How to approach problem• The most logical and efficient means of

addressing? • Most economical?

• Questions and answers

Update from Ohio State!

• New Theriogenology section• Dr. Carlos Pinto• Dr. Marco DaSilva

Update from Ohio StateUpdate from Ohio StateAlpaca embryo transfer program

Goals of the Program:

-Advance knowledge of Alpaca reproductive

physiology through basic and applied research

-Promote development of assisted reproductive

techniques for alpacas

-Increase reproductive efficiency and accelerate

genetic gain of the herd

Areas of Potential Research:

-Protocols for Superovulation

-Maternal Recognition of Pregnancy (ET time)

-Embryo Cryopreservation

-Endocrinology of Pregnancy

Update from Ohio StateUpdate from Ohio StateAlpaca embryo transfer program

Alpaca embryo – Inner cell mass

Pharmacokinetics and bioavailability of florfenicol in Alpacas

Introduction• Florfenicol has been studied

extensively in cattle

• Florfenicol commonly used empirically in camelids – pharmacologic data have never been established in this species

• Goal: to determine the disposition and bioavailability of florfenicol after IV, SQ, and IM dosing

Study design

• 3 – way crossover design with a minimum of 2 weeks wash-out in between studies

• IV dose, IM dose, SQ dose

• 20 mg/kg florfenicol based upon current weights of each study subject

• 8 Male alpacas• 1.5 to 7 years of age

Study day preparation - IV• Accurate body weight

• Place jugular catheter into each vein

• Nuflor administered IV based upon weight in one jugular catheter

• Samples drawn from contralateral catheter

• Centrifuge to obtain serum• Stored in cryovials in duplicate

Study day preparation – IM,SQ

• Same as IV except one catheter placed for sampling

Sample Analysis• Samples prepared for analysis using solid phase

extraction (SPE)

• Analyte quantitation by HPLC UV224 nm

• Samples reconstituted in mobile phase in brown glass vials after concentration and drying (50 μL injections)

• Complete standard curves were simultaneously extracted from blank alpaca serum on each day of analysis

• Unknown sample concentrations were determined by linear regression analysis of known standard concentrations

Method characteristics

• Sample recovery >85% over range of concentrations tested

• Between and within day accuracy was <10%

• Between and within day precision was <10%

• Limit of quantitation – 0.05 μg/mL

Pharmacokinetic analysis• Individual (IV) plasma concentration versus

time data were modeled using compartmental analysis (1, 2, 3 compartments; Topfit 2.0).

• Most appropriate model was selected base upon the Akaike Information Criterion (AIC) and correlation coefficient (r2)

• 2 Compartmental model provided best fit to data

• Data was described in tabular form as median and range

Parameter Units Median Range

A1 (A) μg/mL 110 52 – 275

λ1 (α) hr-1 4.48 2.57 – 14.8

A2 (B) μg/mL 7.6 4.7 – 31

λ2 (β) hr-1 0.367 0.099 – 1.04

k31 hr-1 1.44 0.33 – 4.66

k1e hr-1 1.32 0.54 – 2.0

k13 hr-1 2.0 0.89 – 9.77

MRT hr 2.56 0.86 – 7.36

T½ (elim) Hr 3.75 1.97 – 6.95

Vss L/kg 0.525 0.25 – 2.54

CL mL/min/kg 5.6 1.6 – 6.2

AUC μg*hr/mL 59.8 54 – 208

Tmax hr 0.00016 0.00011 – 0.014

Cmax μg/mL 109 52 - 275

Serum concentration-time curve IV – Florfenicol (20 mg/kg)

Time (hr)

0 5 10 15 20 25

Ser

um

flo

rfen

ico

l co

nce

ntr

atio

n(

g/m

L)

0.01

0.1

1

10

100

1000

Florfenicol IV 20 mg/Kg0.5 g/mL

Concentration-time curves IM and SQ – Florfenicol (20 mg/kg)

Time (hr)

0 5 10 15 20 25

Ser

um

flo

rfen

ico

l co

nce

ntr

atio

n(

g/m

L)

0

1

2

3

4

5

6

7

Florfenicol IM 20 mg/KgFlorfenicol SQ 20 mg/Kg0.5 g/mL

Conclusions• There were no adverse reactions to the

administration of florfenicol intravenously

• Bioavailability low (~11-40%)

• Half-life of elimination is approximately 4 hr post dosing

• Serum concentrations of florfenicol remain above 0.5 μg/mL for at least 15 h post dosing for all 3 routes

• Peak concentrations of florfenicol after IM dosing are higher and less variable than after SQ dosing.

• Administration of florfenicol once daily should provide adequate serum concentrations for most susceptible bacteria

Acknowledgements

• Dr. Jeff Lakritz

• Dr. Andy Niehaus

• Dave Frederick

• Columbus State Technical Students

• Students and Technicians of The Ohio State VMC

Mycoplasma haemolamae

Transmission, virulence, and in vitro culture

Morris Animal Foundation grant funded

Introduction• A hemotropic parasite that attaches to the

membrane surface of red blood cells

• Originally described and classified as an Eperythrozoon-like organism

• Reclassified to Mycoplasma along with M. suis and M. wenyonii (swine and cattle)

• Based on similarity of the 16S ribosomal RNA gene• PCR testing is useful for parasite detection and

isolation of DNA for sequencing

• Colostral antibodies provide immunity for many diseases and parasites – also for M. haemolamae?

Acridine orange staining

AO stains nucleic acid. Mycoplasma has nucleic acid and stains bright yellow

Clinical and Subclinical Disease• Majority of infected animals show no

signs of disease

• Signs of disease are often nonspecific• Lethargy• Decreased appetite• Fever

• Variable degrees of anemia

• Life threatening disease• Severe anemia• Marked hypoglycemia

Transmission• Poorly understood

• Suspected transplacental transmission

• Potential transmission of pathogens via colostrum

• Biting insect vectors

Study Goals

1. To determine if in utero or colostral transmission was a significant route of infection by Mycoplasma haemolamae for neonatal crias.

2. To identify the presence of colostral antibodies, determine if they affect immune status, and evaluate the relationship between dam parasitemia and colostral antibody excretion.

3. Develop an in vitro culture for ongoing studies

Study Design• Sample collection

• 56 dam/cria pairs identified from a single alpaca farm

• Samples obtained• Blood from dam at parturition (0 hr)• Blood from cria at parturition (0 hr)• Colostrum from dam at parturition (0 hr)• Blood from cria after colostrum

ingestion (48-72 hr)• IgG levels checked at birth and post

colostrum

Sample Processing• DNA extraction

• PCR amplification

• Gel electrophoresis of PCR products

• Positive and negative controls

Colostral Antibodies• Several IFA slides prepared with

parasitemic blood sample

• Colostral samples were diluted 1:10 and 1:100 and incubated on the slides

• A goat anti-llama antibody for flurochrome label incubated with slides

• Antibodies are present if RBCs have areas of fluorescence under UV light microscopy

Culture

• Parasitemic animals identified and samples taken and cryopreserved for ongoing studies and culture maintenance

Initial Results and Conclusions

• Transplacental transmission is uncommon but does occur

• Colostral transmission has not been demonstrated

• Antibodies to M. haemolamae are present in colostrum and seem to provide immunity to the parasite

• Further data analysis and publication pending

Acknowledgements

• Dr. Antoinette Marsh

• Dr. Jeff Lakritz

• Dr. Paivi Rajala-Schultz

• Dr. Josh Daniels

• Jackie Daleccio

Stifle Arthroscopy in Camelids

Study Goal and Design• Arthroscopy is commonly performed in

human, canine, and equine patients

• Minimally invasive approach• Decreased morbidity• Excellent joint surface visualization

• Improvement in diagnosis and therapeutic intervention for stifle pathology

• No previous reports in SAC

Joint Model• PMMA injected

into stifle joint

• Soft tissues removed using concentrated NaOH and hypochlorite solution

• Joint anatomy better defined

Stife Arthroscopy

Selected Cases• Llama with severe OA

• Multiple osteochondral fragments• Possible cruciate rupture – unsure

based on exam and radiograph findings• Very heavily conditioned

• Alpaca with patellar fracture• Small basilar fragment• Minimal joint disease• Otherwise healthy

Acknowledgements

• Dr. Andy Niehaus

• Dr. Elizabeth Santschi

• Dave Frederick

Pharmacokinetics of Midazolam in South American Camelids

Anesthesia and Farm Animal Sections Collaborative Project

Dr. Pam Fry, Dr. Turi Aarnes,

Dr. John Hubbell, Dr. Jeff Lakritz

Introduction• Midazolam classified as a

benzodiazapene

• Uses• Sedation• Anesthetic induction

• Less cardiovascular and respiratory depression as compared to diazepam

• No pharmacokinetic studies have been performed in camelids

Study Design• 6 alpacas

• 3 intact males and 3 intact females

• Route of administration• IM and IV• Crossover study design

• Drug levels measured using HPLC

Results Pending• IV and IM show effect at the dosage

administered• IV effect faster onset and more

profound• Subjective findings indicate significant

sedation• No major side effects have been noted

• Drug bioavailability curves are pending

Future Camelid Research at Ohio State• Dental disease in llamas and alpacas

• Tooth root abscesses occur commonly in llamas and alpacas

• Other ruminants seem less predisposed

• Identification of feed, forage, or other husbandry practices that may predispose to dental disease

• Current plan includes surveying owners in conjunction with retrospective case analysis from hospitalized patients

Update from Ohio State!

• International Camelid Health Conference for Veterinarians

• March 2012 (2nd or 3rd week)• Camelid owners/breeders conference• Veterinary speakers from across the

USA and world

Buckeye Alpaca Show sponsors ICHC speakers

• ICHC for DVM sponsorship by the Buckeye Alpaca Show and OABA?

• Look to OABA and OSU CE website for more details coming soon

ICHC for DVM meeting – tentative schedule• Modules of expertise in

cohesive blocks • Individual

presentation• Summary statements• Discussion/questions

• Reproduction• Medical Diseases• Surgical Diseases• International presence

ICI update –

• Vets only forum• Duplicated by AOBA, NAAF(?)

• Veterinary videos – Collaboration with ARI

• Reference Library• Updated references

• Endowment• Value ~ 700K

Parasites and Camelids –

Should We Be Worried?Becky Pentecost, DVM

The Ohio State University

Veterinary Medical Center

Hospital for Farm Animals

The top 10 reasons camelids develop GI disorders –

• 1. Parasites

• 2. Parasites

• 3. Parasites

• …………..

Why discuss parasites? • What is your parasite management

program?• How developed?

• What is the goal of your parasite management program?

• What is the cornerstone of your parasite management program?

• What would you do if tomorrow, none of the deworming agents worked on any parasites?

Parasitism in SAC

• Camelids can survive pretty well on low quality feed

• Increased digestion and fermentation of even coarse hay

• Water conservation is impressive• Camelid parasites in North America

• SAC have evolved without some of the parasites commonly encountered in NA

Common Nematode ParasitesGI Tract Location ParasitesC3 Haemonchus contortus,

Trichostrongylus, Ostertagia, Camelostrongylus, Teladorsagia, and Marshallagia

Small Intestines Nematodirus, Cooperia, Trichostrongylus

Large Intestines/Cecum Trichuris, Capillaria, and Oesophagostumum spp

Life Cycle Review for Trichostrongyle-types

Infective larvae are ingested during grazing

Eggs hatch, larvae develop to L3 in soil and manure

Eggs passed onto pasture in manure

Adult nematodes in the digestive tract of camelids lay their eggs.

Cestode Parasites• Moniezia most common cestode

• Generally not a primary pathogen

• Severe infections can cause generalized signs of unthriftiness

• Treatment: Fenbendazole, albendazole, or praziquantel

Protozoal Parasites• Coccidia – Eimeria spp.

• Six species reported – 4 are common• E. punoensis, E. alpacae, E. lamae, E.

macusaniensis

• Direct fecal-oral transmission

• Healthy adults rarely show signs of clinical disease

• Juveniles more susceptible• Immunity develops after exposure

Coccidia• Damage intestinal epithelial mucosal

cells

• Diarrhea may remain after clearing infection

• Coccidiostats during times of high stress may help prevent outbreaks

• Amprolium• Sulfadimethoxine• Ponazuril• Toltrazuril

E. mac – A Big Deal?• First reported in the US in 1988

• Slightly longer prepatent period than other Eimeria spp

• Widespread geographic distribution

• Can be subclinical carriers/shedders

• Can cause serious disease/death

• Evaluate for signs of infection• Protein levels of particular concern• Ponazuril generally accepted treatment• Toltrazuril becoming more popular

Indications of GI Parasitism• Often nonspecific

• Unthrifty appearance• Anorexia• Slow or stunted growth• Weight loss / Lower BCS

• Diarrhea

• Pale mucous membranes

• Edema (submandibular or ventral)

Fecal Exams• Preferred methodology

• Concentrated sugar solution (1.27 SG)• Centrifugation• 30-60 minute flotation time

• Qualitative vs. Quantitative Analysis

• Fecal Egg Count Reduction Test (FECRT)

Other Diagnostics?

• PCV/TP

• CBC

• Blood transfusion may be required in severely anemic animals as a stabilization measure

Anthelmintics• Accurate body weights

• Deworming history• Timing• Fecal counts• Effectiveness of previous dewormers

• Targeted treatment

• Resistant parasites

• Limited dewormers available – no new ones in sight…

Available DewormersClass Drug Dosage Route

Avermectins Ivermectin 1.5 mL/100 lbs SQ

Doramectin 2.0 mL/100 lbs SQ

Cydectin

Benzamidazoles Fenbendazole 9 mg/lb Oral

Albendazole 5.5 mg/lb Oral

Imidazothiazole derivative

Levamisole 4 mg/lb Oral

Depolarizing neuromuscular blocking agent

Pyrantel pamoate 8 mg/lb Oral

Antiprotazoal/Coccidiostats

Ponazuril 9 mg/lb Oral

Toltrazuril

Amprolium

Sulfadimethoxine

Deworming Conundrum• Meningeal worm

• Deworming recommendations to prevent infection• Ivermectin• Doramectin• Treat monthly

• What is the downside to this treatment pattern?

• Resistant GI parasites

Monthly deworming with Ivermectins – How effective against whipworms? • Ivermectins are of highly variable efficacy against

Trichuris spp. in swine. • Riviere J, Papich M, Veterinary Pharmacology and

Therapeutics 9th Ed. 2009. pp. 1131

• Ivermectin at higher doses for dogs and cats• Riviere J, Papich M, Veterinary Pharmacology and

Therapeutics 9th Ed. 2009. pp. 1131

• Parasite fecundity – Low

• Eggs high density; must use appropriate centrifugation techniques

• Camelids highly susceptible to whipworm infestation

Monthly deworming with Ivermectins – How effective against mites?

• Reportedly highly effective against Sarcoptic mange, Psoroptic mange

• Effective against sucking lice• Chorioptic mange?

• Biology of this parasite?• May spend weeks off of host (environment)• Treatment with Doramectin or Ivermectin

• G.L. D’Alterio, Vet. Parasit. 2005; 130 (3-4): 267-275• Fipronil application (topical, spray)

FECR tests – Goats 2001Anthelmintic EPG (2 wk post) FECR % (95% CI)

Control 3827

ABZ 1450 62 (35-78)

Dora 917 76 (39-91)

FBZ 3460 10 (0-42)

IVM 844 78 (5-95)

LEV 335 91 (81-96)

Morantel 1990 48 (11-70)

MOXI 0 100 (100)

ABZ + IVM 445 88 (74-95)

Terrill TH, et al., 2001; Vet. Parasitol. 97:261-268

FECR tests – Goats 2003• ABZ resistance on 14/15 farms• IVM resistance on 17/18 farms• LEV resistance on 6/18 farms• MOXI resistance on 1/18 farms• Resistance to multiple drugs

• 14/15 farms (ABZ, IVM)• 5/15 farms (ABZ, IVM, LEV)

• FEC reduction (%)• ABZ 67, IVM 54, LEV 94, MOXI 97

• H. contortus and Trichostrongylus most common isolated larvae

Mortenson LL, et al., 2003; JAVMA 223:495-500

FECR tests – Sheep and Goats• Haemonchus resistance

• 45/46 farms – BZD• 25/46 farms – LEV• 35/46 farms – IVM • 11/46 farms – MOXI

• Resistance to all 3 classes• 22/46 farms

• Resistance to all 3 classes + MOXI• 8/46 farms

• H. Contortus and Trichostronylus most common identified parasites

Howell SB, et al., 2008; JAVMA 233:1913-1919

Parasite resistance in SAC?

• What is the incidence of resistant nematodes in SAC?

• Gillespie RM et al., 2010; Vet. Parasit. 172:168-171• Resistance to IVM (Llama, Alpaca farms)• Resistance to FBZ (Llama farms)• Resistance to MOX (Llama farm)• No Resistance to LEV on any farm tested

Parasite resistance and anemia

• Seeing increased incidence of parasite problems

• Many of these present with severe anemia

• Haemonchosis?

H. Contortus – C3, alpaca

Blood loss ?

• Blood volume in llamas 63 ± 4 mL/Kg, alpacas 72 ± 5 mL/Kg

• Blood volume 9L (llama), 4.9L (alpaca)

• Anemia hemorrhagic in nature• Adult worms suck blood for 12 min, 7 min

continued bleeding after worm detachment

• Sheep carrying 3-4k female worms lose up to 150-200 mL whole blood/day

Moore DM, Vet. Hematology; 5th Ed. Pp. 1184-90Le Jambre LF, Int. J. Parasitol, 1995; 25:269-73Rowe JB, et al., British J Nutrition 1988; 59:125-39

Fecal ooccult blood –

The Predicament – Death of your animals

• Monthly deworming for meningeal worm

• Selection for resistant nematodes

• Overdosing dewormers

• Anemia and death

Building a parasite program

• What things can you incorporate?• 1. Camelid behavior• 2. Pasture management • 3. Biological controls

Peculiarities of camelids – • Dung piles

• Most important aspect of camelid behavior relative to GI parasitism management

• Overcrowding

Map of property – Why?

Integrated parasite management

• Clean or safe pastures • Pastures not grazed for 6-

12 mos • Pastures grazed by other

species• Pastures where hay is

removed• Pastures rotated with

harvested crops• Tilled land put into pasture• Pastures rested for varying

time period• 60 -120 day between grazing• Improved pasture outweighs

parasite burdens

Grazing strategy• 80% of worm burden in lower 2” of grass

• Grazing taller grass will limit exposure

• Browsing

• Wait until dew or rain dries• Turn out later in day

• Multi-species grazing

• Alternative forages• Chicory• Sericea lespedeza• Birdsfoot trefoil

Other Management Techniques• Feeding off the ground (bunks)

• Eliminating standing water

• Regular removal of dung piles and pen sanitation

• Biosecurity - Quarantine incoming animals

Anemia in South American camelids –

• Gastrointestinal parasitism• Primarily Haemonchus

• Blood borne pathogens• Mycoplasma haemolamae

• Toxic• Red Maple

• Neoplasia• Lymphoma

Review of recent anemia cases from OSU VMC

• 46 Animals (35 Alpaca, 11 Llama)

• 33 female, 13 male; 4.2 ± 3.5 yrs• 41 heavily parasitized, 2 obstetrical complications, 1 peritonitis, 1 tetrology of fallot.

Iron deficiency anemia – • Microcytic, hypochromic, reduced

MCHC, hypoferremia, reduced serum ferritin conc, decreased % transferrin saturation and normal to increased TIBC

• Moderate anemia, PCV 18-22, RBC# normal, hemoglobin ½ normal, microcytic, low MCHC

• Morin DE et al, Vet Pathol. 1992; 400-4

Iron deficiency anemia

• 2/3 of body iron is present in RBC

• Bone marrow, spleen, liver

• Serum iron drops first, followed later by BM decrease and change in RBC morphology (hypochromasia, microcytosis)

• Vitamin B12 deficiency• Macrocytic anemia

From: Smith BP, 2009; 4th Ed., 2009

Total plasma protein and red cell parameters – presentation

Analyte Mean ± sd Range Median Reference

TPP (Gm/dL) 5.2 ± 0.72 3.7 – 6.6 5.3 5.4 – 7.2

HCT (%) 9.9 ± 5.3 4 – 25 8 24 – 35

HgB (Gm/dL) 4.2 ± 2.3 1.6 – 11 3.6 9.1 – 16.2

RBC (x 1012/L) 4.2 ± 2.5 1.4 – 10.5 3 8.8 – 15.4

MCV (fL) 25 ± 6 12.7 - 44 24 21 - 30

MCH (pg) 10 ± 2 6.5 – 16 10 7.7 - 13

MCHC (g/dL) 42 ± 5.7 35.7 - 48 41 39 - 46

NRBC (ABS) 28 ± 47 0 - 257 15 0 – 0

NRBC

(#/100 WBC)4.8 ± 8.2 0 - 42 2 0 – 1

RBC abnormalities - ?

Polychromatic, hypochromatic, dacryocytes

Serum Fe, TIBC, UIBC in anemic Alpacas

Analyte UnitsAnemic

(median)Range

Normal

(median)Range

Serum

Feμg/dL 43 ± 42*

(22)2 – 121

95 ± 35

(96)37 - 168

TIBC μg/dL261 ± 65

(266)172 – 363

248 ± 44

(242)186 – 339

UIBC μg/dL

218 ± 71

*(209)

70 - 309153 ± 51

(164)61 – 230

Data from 13 anemic and 13 normal Alpacas*Indicates significant difference from normal using t-test(P<0.05); power with α = 0.05 are 0.915, 0.67 respectively

“Otis May” – Presentation

• Down, weak, pale

• No milk in udder

• Cria is hungry and trying to nurse

• Ocular membranes

Case Management• White mucous membranes

• PCV – 7%

• TP – 5.0 g/dL

• CBC indicates regenerative response (NRBCs elevated)

• Mild derangements on biochemical profile

• Blood transfusion – 1-2 units whole blood

• Iron and Vitamin B12 supplementation

• Fecal examination, PCR for M. haemolamae, etc.

Questions?