559597

research-article2014
VDIXXX10.1177/1040638714559597Tungiasis in cattleMarin et al.

Brief Communication

Journal of Veterinary Diagnostic Investigation

Pathology and diagnosis of proliferative and 2015, Vol. 27(1) 80­–85

© 2014 The Author(s)
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DOI: 10.1177/1040638714559597
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penetrans infestation in cattle

Raul E. Marin, Robin Houston, Alicja Omanska-Klusek, Ana Alcaraz,

Jorge P. Garcia, Francisco A. Uzal1

Abstract. Tunga penetrans is the smallest biting flea known. In cattle, infestation by T. penetrans (tungiasis) typically
affects the skin of the distal legs, udder, prepuce, and perianal area. A detailed clinical and pathologic description of bovine
tungiasis, together with electron microscopy and molecular diagnostics to establish the identity of the parasite are described.
Ninety percent of the cows and heifers and 80% of the bulls in a herd in northwest Argentina had proliferative and ulcerative
skin lesions affecting the coronary band, interdigital space, heels, and rudimentary toes of the fore and/or rear limbs, teats, and/
or prepuce. These proliferative lesions had multiple large cavities filled with hemorrhagic fluid, necrotic debris, and Tunga spp.
parasites. Histologically, the skin showed diffuse papillary epithelial hyperplasia with severe orthokeratotic hyperkeratosis,
and it was multifocally ulcerated and inflamed. Multifocally, sections of arthropod parasites were observed embedded in the
epidermis and dermis with the posterior end toward the surface. Images of wet mounts and scanning electron microscopy of the
parasite showed morphologic characteristics compatible with Tunga spp. Polymerase chain reaction followed by sequencing
of the cytochrome c oxidase subunit II and the internal transcribed spacer region indicated 99% homology to published
T. penetrans sequences. Tungiasis should be considered as a differential diagnosis for proliferative lesions in skin of cattle.

Key words: Cattle; dermatitis; Tunga penetrans; tungiasis.

Proliferative lesions of the skin and adnexa in cattle can be
associated with various etiologies including bacterial, viral,
fungal, and parasitic agents.4 Insects, such as Tunga pene-
trans, have also been described as causing proliferative
dermatitis in cattle, although information about the pathol-
ogy of the lesions produced by this parasite in cattle is very
limited.2,9,12
The genus Tunga includes 13 species of sand fleas, of
which T. penetrans is the most frequently reported, parasit-
izing several wild and domestic mammalian species includ-
ing human beings, cattle, dogs, cats, pigs, goats, sheep,
monkeys, wild rodents, coatis, and armadillos.6,13 The dis-
ease produced by the female sand flea of the genus Tunga is
known as tungiasis. T. penetrans (also known as sand flea,
nigua, pique, chigger, or bug of the foot) is the smallest
known biting flea (approximately 1 mm diameter).3 The
gravid female penetrates the skin of the host and once in the
skin undergoes significant hypertrophy reaching a size of
up to 1 cm.5 It breathes, defecates, expels the eggs, and
stays in contact with the air via its abdominal cone, leaving
an opening of approximately 250–500-µm diameter in the
skin, which can be a port of entry for microorganisms.5 No
drug treatment is known to be effective to eliminate bur-
rowed sand fleas, although insect repellents seem to help in
preventing infestation.3 The treatment of both human and
animal tungiasis consists mainly of surgical extraction of
the fleas, followed by cleansing and topical antibiotics to
prevent secondary infections. Although topical ivermectin,
metrifonate, and thiabendazole have been reported to be
effective in killing the fleas and facilitate their manual
removal, these treatments do not remove the flea from the
skin and do not provide relief for the painful Tunga spp.
lesions.1,7,8 Preventive fumigation of problem fields and
pens with pyrethroid substances at the beginning of the dry
season seems to help in preventing infestations (authors’
unpublished observation).
In human beings, tungiasis by T. penetrans is endemic in
several areas of South and Central America, and Africa.14
Isolated cases of this disease have also been reported in India
and Italy.6,14 Originally T. penetrans existed only in the
From private practice, Jujuy, Argentina (Marin); Veterinary Medical
Teaching Hospital (Houston), Department of Medicine and Epidemiology
(Omanska-Klusek), California Animal Health & Food Safety Laboratory,
San Bernardino branch (Garcia, Uzal), School of Veterinary Medicine,
University of California–Davis, Davis, CA; and College of Veterinary
Medicine, Western University of Health Sciences, Pomona, CA (Alcaraz).
1
Corresponding Author: Francisco A. Uzal, California Animal Health
& Food Safety Laboratory, San Bernardino Branch, 105 W. Central
Avenue, San Bernardino, CA 92408. fuzal@cahfs.ucdavis.edu

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 Tungiasis in cattle
Americas from where it propagated to other parts of the
world.3 Both in human beings and animals, tungiasis is
mainly associated with sandy soils and dry weather, but it
has also occasionally been seen in rainforests.3
Bovine tungiasis has been described in several South
American countries with most reports originating from Bra-
zil.10,12 The disease in cattle can be produced by 3 species of
Tunga (i.e., Tunga penetrans, Tunga trimamillata, and Tunga
hexalobulata),2,10 although T. penetrans is by far the most
frequently reported species of Tunga associated with bovine
tungiasis.
Tungiasis by T. penetrans in cattle usually affects the skin
of the distal feet, udder, prepuce, and perianal area. Preva-
lences between 2.5% and 68% have been described in bovine
herds.2,12 Lesions in bovine tungiasis are painful, pruritic,
and often secondarily contaminated with opportunistic bac-
teria.12 Treatment is rarely applied to cattle and typically the
outcome of infection is culling due to severe lameness, sec-
ondary infections, and the drop in production associated with
these lesions.
Previous reports of bovine tungiasis have described only
the epidemiological and clinical characteristics of the dis-
ease; while the identification of Tunga spp. has been tradi-
tionally limited to morphological features alone.2,12 The
clinical and pathological changes produced by different spe-
cies of Tunga spp. are very similar to each other, and deter-
mination of the species present cannot be achieved by these
features alone. The current study presents a detailed patho-
logic description of bovine tungiasis together with the use of
electron microscopic findings and molecular techniques that
confirmed the identity of the parasite.
In 2007, the owner of a breeding herd of Brangus cross
cattle (n = 1,430, including cows, heifers, steers, bulls, and
calves) in the province of Salta, northwest Argentina,
reported a high incidence of digital lesions, mainly in the
adult cattle. The farm was visited by one of the authors (R
Marin), and all the cows and heifers (n = 520) and bulls (n =
30) were examined clinically with special emphasis on feet,
udders, and in the case of the bulls, prepuce. Acute clinical
signs consisted of intense pruritus, pain, licking, and swell-
ing of affected areas (mostly feet and teats), and obstruction
of the teat canal. The latter led to severe reduction in milk
production and mastitis, which in turn was responsible for
loss of condition and death of a large number of calves. Ani-
mals that had been affected for several days presented hoof
deformities, anorexia with severe loss of condition, lame-
ness, and marked reduction of reproductive indexes. The
bulls showed marked loss of libido.
An adult cow with feet and teat lesions similar in nature
and severity to those seen in most animals of the herd was
euthanized, and a full necropsy, including thorough gross
examination of the skin, with particular emphasis on limbs
and udder, was performed. Samples from leg and teat lesions
were collected in 10% buffered formalin (pH 7) and pro-
cessed routinely for the production of 4-µm thick hematoxy-
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81
lin and eosin–stained sections. Selected sections of skin were
also stained with Steiner, Gram, and an indirect immunoper-
oxidase technique for Treponema spp. using a commercial
kita according to previous descriptions.11 For the latter, a rab-
bit polyclonal anti–Treponema spp. antibodyb was used as a
primary antibody. Skin from the interdigital space of a cow
with papillomatous digital dermatitis and from the interdigi-
tal space of a normal cow, were used as positive and negative
controls, respectively.
Samples from the legs and teat lesions from the eutha-
nized cow were aseptically collected, inoculated onto 5%
blood agar plates, and incubated aerobically or anaerobically
for 48 hr. Colonies were examined on Gram-stained smears
and finally identified by conventional biochemical tests.
Direct smears of fine-needle aspirates from the leg lesions
were also examined by dark field microscopy.
Fleas from the environment and from the skin of several
affected animals were collected, fixed initially in 70% etha-
nol, and postfixed in Karnovsky fixative in 0.1 M sodium
phosphate buffer (Sorenson), then washed using 0.1 M
sodium phosphate. Dehydration was accomplished in
increasing concentrations of ethanol through 100% ethanol
and critical point dried.c The fleas were mounted on alumi-
num stubs and sputter coated with gold using a commercial
coater.d The samples were viewed on a scanning electron
microscope (SEM),e and digital images were obtained.
DNA from 3 fleas was extracted using a commercial blood
and tissue kitf according to the manufacturer’s instructions.
Two different sets of primers specific for T. penetrans were
designed, one to detect cytochrome c oxidase subunit II
(COII; accession no. DQ 844706.1) and another to detect
5.8S ribosomal RNA gene internal transcribed spacer (ITS;
accession no. DQ844725.1). The following COII primers
with expected amplicon size of 468 bp were used: forward
5′-AATTTACTCACCGAATATTAATAGAAAGTCAA-3′,
and reverse 5′-CTATGATTTGCTCCACAGATTTCTG-3′.
The following ITS primers with expected amplicon size of
396 bp were used: forward 5′-CTAATTGCGCGTCAACAT
GTG-3′ and reverse 5′-AAGCGTGGAGGTTTC GAGTTC-
3′. The PCR amplification was carried out using commercial
kitg consisting of 5 μl of 10× Advantage 2 buffer,g 1 μl of 10
mM deoxyribonucleotide triphosphate mix, 1 μl of each
primer, 20 μl of extracted DNA, 21 μl of diethylpyrocarbon-
ate-treated water, and 1 μl of 50× Advantage 2 polymerase
mixg for a total reaction volume of 50 μl. The following
cycling conditions were used: 94°C for 1 min, 25 cycles of
94°C for 15 sec, 55°C for 15 sec, and 70°C for 45 sec, follow-
ing with a final elongation of 70°C for 5 min and a 4°C for-
ever hold step. The PCR products were run on 1.8% agarose
gel with addition of EZ VISION dye,h and the expected sizes
of bands were noted. Each band was cut out, purifiedi accord-
ing to manufacturer’s instructions, and submitted to the
sequencing facility of the University of California–Davis.
Ninety percent of the cows and heifers (468/520) and 80%
of the bulls (24/30) had proliferative skin lesions affecting 1 or
82
Marin et al.
Figure 1. Cow. Distal limb with proliferative and ulcerative
dermatitis and coronitis due to Tunga penetrans.
Figure 2. Cow. Udder with proliferative and ulcerative
dermatitis due to Tunga penetrans.
more of the following locations: fetlock area, rudimentary
toes, coronary band, interdigital space and heels of the fore
and/or rear limbs, teats, and/or prepuce. Proliferative lesions
in most animals were ulcerated and bleeding.
In the euthanized adult cow, 4 legs and 4 teats were
affected. Proliferative and hemorrhagic lesions of varying
sizes (3 cm × 2 cm × 2 cm to 15 cm × 10 cm × 6 cm) were
present on all 4 feet distal to the fetlock joint, including the
coronary band and interdigital space (Fig. 1), and the teats
(Fig. 2); there was also abnormal hoof growth (Fig. 3). Cut
section of the leg and teat lesions revealed multiple large
cavities that contained hemorrhagic fluid, necrotic debris and
Tunga spp. parasites. Subcutaneous edema extended distally
from the carpi and tarsi to the coronary band. Lesions in the
teats were similar but less severe. No other significant gross
abnormalities were observed in the rest of the carcass.
Histologic findings were similar in leg and teat lesions.
The epidermis showed diffuse papillary hyperplasia with
severe orthokeratotic hyperkeratosis (Figs. 4, 5). Multifo-
cally, there was deep chronic ulceration covered by large
crusts composed of extravasated red blood cells, lympho-
cytes, plasma cells, macrophages, neutrophils, eosinophils,
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Figure 3. Cow. Distal limb with proliferative and ulcerative
dermatitis, and coronitis due to Tunga penetrans. Notice deformed
hooves.
Figure 4.  Cow. Subgross section of skin with prominent
orthokeratotic hyperkeratosis. Observe an intradermal and
epidermal section of an arthropod compatible with Tunga
penetrans, with posterior end toward the surface. The parasite
shows a thick cuticle, prominent hypodermic cells lining the body
cavity, distended digestive tract sections, and posterior end with
chitin. Hematoxylin and eosin (HE). Bar = 100 μm. Inset: higher
magnification of the external parasite wall (right) and dermal host
tissue (left) showing neutrophilic and eosinophilic infiltration. HE.
Bar = 20 μm.
fibrin, cell debris, and bacterial rods and cocci. The dermis
showed diffuse infiltration by lymphocytes, plasma cells,
macrophages, eosinophils, and neutrophils (which was most
severe surrounding parasites), fibroplasia with proliferating
blood vessels oriented perpendicular to the surface (granula-
tion tissue), and pockets of necrosis and neutrophilic infiltra-
tion, which included both viable and dead neutrophils.
Hematoxylin and eosin– and Steiner-stained sections of skin
showed superficial invasion of mixed bacteria, which multi-
focally invaded the superficial dermis. This bacterial popula-
tion was composed by a mixture of Gram-positive and
-negative organisms. Immunohistochemical staining for
Treponema spp. was negative in all sections examined.
 Tungiasis in cattle 83

Figure 5.  Cow. Higher magnification of the posterior end of

Tunga penetrans in the skin showed in Figure 3. The image shows
the thick basophilic cuticle of the exoskeleton (Ex), distended
intestinal loops (G), trachea (T), developing eggs (E), pale-staining
areas (Psa), and external orifice (Eo). Hematoxylin and eosin. Bar
= 40 μm.
Figure 6. Light micrograph of a non-gravid female Tunga
penetrans in left lateral recumbency. T: tibia of left hind leg; GD:
genital depression. Wet mount. Bar = 200 μm.

Multifocally, sections of arthropod parasites were embed-

ded in the epidermis and dermis with the posterior end ori-
ented toward the surface (Figs. 4, 5). The parasites were
approximately 2 mm × 3 mm in length, with a thick body
wall lined by a prominent hypodermal layer and cuticular-
ized structures on the external surface. The head presented a
well-developed lacinia and epipharynx. Within the body cav-
ity there were myriad, approximately 100 µm diameter eggs
at various stages of development. The eggs were round and
solid or targetoid (Fig. 5). Long eosinophilic muscle fibers
with central elongated nuclei, and tubular transverse sections
of intestine lined by prominent epithelium, trachea, and pale-
staining areas were also seen scattered within the parasite
body wall (Fig. 5).
Bacterial cultures of leg and teat skin samples produced
almost pure growth of Pseudomonas spp. Spirochetes were Figure 7.  Light micrograph of an adult male Tunga penetrans
not seen on direct smears of fine-needle aspirates from the in left lateral recumbency. T: tibia of left hind leg; CA: copulatory
leg lesions examined by dark field microscopy. apparatus. Wet mount. Bar = 200 μm.
Light microscopy of wet mounts (Figs. 6, 7) and SEM
images (Figs. 8, 9) of male and non-gravid female parasites A diagnosis of tungiasis was made based on the clinical,
showed a relatively flattened head. The genal groove, char- gross, and histologic findings. Light microscopic and SEM
acteristic of T. penetrans, was visible on wet mounts and examination of the parasites allowed identification of the
SEM of both sexes (Figs. 6–9), as was the absence of setae parasite as T. penetrans. Pale-staining areas were seen within
on the medial border of the tibia (Fig. 10). Sexual dimor- the parasite wall. Similar pale-staining areas have been pre-
phism was characteristic of the genus (i.e., the male possess- viously described in the inner and outer portions of the sand
ing a prominent copulatory apparatus and the female having flea’s posterior region of the exoskeleton.14 These pale areas
a distinct genital depression). seem to be unique for T. penetrans, and they are considered
Sequencing results for COII indicated 99% homology to to be useful identifying features. Further confirmation of the
published T. penetrans sequences. There was a 3-bp mis- species as T. penetrans was provided by sequencing of the
match. The sequence for the ITS region of T. penetrans also COII and ITS, both of which showed 99% homology to the
indicated 99% homology to the known published sequences published sequences of this parasite. Even though both frag-
of this parasite with a 5-bp mismatch. ments had a few base pair mismatches to the currently

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84
Marin et al.
Figure 8.  Nongravid female Tunga penetrans in left lateral
recumbency. GD: genital depression; A: antenna; E: eye; GG:
genal groove; LP: labial palps. Scanning electron microscopy.
Bar = 200 μm.
Figure 9. Male Tunga penetrans in left lateral recumbency.
CA: copulatory apparatus; A: antenna; E: eye; GG: genal groove;
MP: maxillary palps. Bar = 200 μm.
published sequence data, the primers were designed on areas
that would amplify regions conserved to T. penetrans spe-
cies, and sequenced fragments significantly differed from T.
trimamillata, which allowed ruling out the latter. Other
causes of proliferative dermatitis were ruled out based on
clinical, gross, and microscopic examination, as well as
immunohistochemical staining for Treponema spp., and bac-
terial cultures.
Although 13 species of Tunga have been described, only
3 of them (i.e., T. penetrans, T. trimamillata, and T. hexa-
lobulata) have been described in cattle, with the first one
being most frequently seen.2,10 Differentiation of the 3 spe-
cies of Tunga that affect cattle based on morphology of the
parasite alone is difficult and frequently inaccurate. The use
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Figure 10. Light micrograph of the tibia of a female Tunga
penetrans. The region between the arrows shows the lack of setae
that distinguishes T. penetrans from Tunga trimamillata. Wet
mount. Bar = 40 μm.
of molecular methods in the current study proved to be a
quick and efficacious method to identify Tunga at a species
level. Identifying fleas to the species level may prove useful
to select appropriate control and treatment measures if differ-
ences in sensitivity and pathogenesis are found between flea
species in the future.
Between 80% and 90% of the animals in the study farm
had proliferative lesions consistent with tungiasis. Similar
prevalences were observed in some neighboring farms
(authors’ unpublished observations). To the best of the
authors’ knowledge, this high prevalence has not been previ-
ously reported in cattle or other animal species. Tunga pen-
etrans thrives in semitropical or tropical climates with sandy
soils, as was present in the study farm. However, a high prev-
alence of tungiasis was not always seen in neighboring farms
with similar conditions. The reason for the high prevalence
of disease seen in the current study is unknown; further epi-
demiological studies are necessary to clarify this issue.
Tungiasis should be considered as a differential diagnosis
for proliferative lesions in skin and adnexa of cattle. Molecu-
lar methods are very useful for rapid identification of the
parasite at the species level. Fleas and/or full thickness skin
samples are required for diagnosis.
Acknowledgements
All SEM work was completed in the Electron Microscopy Labora-
tory, Department of Medical Pathology and Laboratory Medicine,
School of Medicine, University of California at Davis. The authors
thank Ms. Patricia Kysar for her technical assistance and the field
staff of San Jose de Pocoy, Salta, Argentina, for their help in several
ways.
Sources and manufacturers
a. ABC Vectastain kit, Vector Laboratories Inc., Burlingame,
CA.
b. Rabbit polyclonal anti-treponema antibody, CAHFS-Davis,
CA.
c. Critical point dryer: Tousimis 931.GL Autosamdri, Tousimis
Research Corp., Rockville, MD.
d. Pelco auto sputter coater SC-7, Ted Pella Inc., Redding, CA.
 Tungiasis in cattle
e. Philips XL30 TMP, FEI Co., Hillsboro, OR.
f. DNeasy blood and tissue kit, Qiagen NV, Venlo, The
Netherlands.
g. Advantage 2 polymerase kit (catalog no. 639206), Clontech
Laboratories Inc., Palo Alto, CA.
h. EZ Vision dye, Amresco Inc., Solon, OH.
i. QIAquick gel extraction kit, Qiagen NV, Venlo, The
Netherlands.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, author-
ship, and/or publication of this article.
10. Linardi PM, De Alvear DM, Facury Filho EJ: 2013,
References
1. Clyti E, Couppie P, Deligny C, et al.: 2003, Efficacite de la
vaseline salicylee a 20% dans le traitement des tungoses pro-
fuses. A propos de huit observations en Guyane Francaise
[Effectiveness of 20% salicylated vaseline in the treatment
of profuse tungiasis. Report of 8 cases in French Guiana].
Bull Soc Pathol Exot 96:412–414. In French. Abstract in
English.
2. De Avelar DM, Facury Filho EJ, Linardi PM: 2013, A new
species of Tunga (Siphonaptera: Tungidae) parasitizing cattle
from Brazil. J Med Entomol 50:679–684.
3. Feldmeier H, Sentongo E, Krantz I: 2013, Tungiasis (sand flea
disease): a parasitic disease with particular challenges for pub-
lic health. Eur J Clin Microbiol Infect Dis 32:19–26.
4. Ginn P, Mansell P, Rakich PM: 2006, Skin and Appendages.
In: Jubb, Kennedy and Palmer’s pathology of domestic
Downloaded from vdi.sagepub.com at UNIVERSITE DE MONTREAL on July 9, 2015
85
animals, ed. Maxie MG, 5th ed., pp. 553–781. Elsevier
Saunders, Edinburgh, UK.
5. Haddad V Jr, Costa Cardoso JL, Lupi O, Tyring SK: 2012,
Tropical dermatology: venomous arthropods and human skin:
Part I. Insecta. J Am Acad Dermatol 67:331.e1–14.
6. Heukelbach J: 2006, Revision on tungiasis: treatment options
and prevention. Expert Rev Anti Infect Ther 4:151–157.
7. Heukelbach J, Eisele M, Jackson A, Feldmeier H: 2003,
Topical treatment of tungiasis: a randomized, controlled trial.
Ann Trop Med Parasitol 97:743–749.
8. Heukelbach J, Franck S, Feldmeier H: 2004, Therapy of tungi-
asis: a double-blinded randomized controlled trial with oral
ivermectin. Mem Inst Oswaldo Cruz 99:873–876.
9. Leao MA, Da Silva LAF, Soares Fioravanti MC, et al.: 2005.
Dermatite digital bovina: Aspectos relacionados à evolução
clinica [Bovine digital dermatitis: aspects related to clinical
outcome]. Ciencia Animal Brasilera 6:267–277. In Portuguese.
Establishment of Tunga trimamillata (Siphonaptera: Tungidae)
in Brazil. Parasitol Res 112:3239–3242.
11. Read DH, Walker RL: 1998, Papillomatous digital dermatitis
(footwarts) in California dairy cattle: clinical and gross patho-
logic findings. J Vet Diagn Invest 10:67–76.
12. Ribeiro JCVC: 2007, Infestação de Tunga penetrans siphon-
aptera: Tungidae em cascos de vacas leiteiras F1 Holandês-
Zebu [Tunga penetrans infestation Siphonaptera: Tungidae in
hooves of F1 Holstein-Zebu cows]. Arq Bras Med Vet Zootec
59:520–522. In Portuguese. Abstract in English.
13. Sanusi ID, Brown EB, Shepard TG, Grafton WD: 1989,
Tungiasis: report of one case and review of the 14 reported
cases in the United States. J Am Acad Dermatol 20:941–944.
14. Smith MD, Procop GW: 2002, Typical histologic features
of Tunga penetrans in skin biopsies. Arch Pathol Lab Med
126:714–716.