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Dourine: a neglected disease of equids

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Trop Anim Health Prod
DOI 10.1007/s11250-017-1280-1

REVIEWS

Dourine: a neglected disease of equids

Yonas Gizaw 1 & Mulisa Megersa 1 & Teka Fayera 1

Received: 15 September 2016 / Accepted: 15 March 2017

# The Author(s) 2017. This article is an open access publication

Abstract Dourine is a venereal transmitted trypanosomosis June 2011 in Sicily and then just north of Naples, on the
causing a major health problem threatening equines world- Italian mainland (Sidney et al. 2013).
wide. The origin and identification of Trypanosoma T. equiperdum is morphologically identical to other insect
equiperdum within the subgenus Trypanozoon is still a subject vector-transmitted Trypanosoma evansi and Trypanosoma
of debate. Unlike other trypanosomal infections, dourine is brucei which cause surra and nagana, respectively. In many
transmitted almost exclusively by coitus. Diagnosis of dourine regions of the world, these three parasite species occur togeth-
has continued to be a challenge, due to limited knowledge er and current diagnostic tests are unable to differentiate be-
about the parasite and host-parasite interaction following in- tween them (Brun et al. 1998). T. equiperdum differs from
fection. The pathological lesions caused by the diseases are other trypanosomes in that it is primarily a tissue parasite that
poorly described and are observed mainly in the reproductive rarely invades the blood. The trypanosomes, which are present
organs, in the nervous system, and on the skin. Dourine has in the seminal fluid and mucous membranes of the genitalia of
been neglected by research and current knowledge on the the infected donor animal, are transferred to the recipient dur-
disease, and the parasite is very deficient despite its consider- ing sexual intercourse. Parasites then may pass into the blood,
ably high burden. This paper looks in to the challenges in where they are carried to other parts of the body. In typical
identification of T. equiperdum and diagnosis techniques with cases, this metastatic invasion gives rise to characteristic cu-
the aim to update our current knowledge of the disease. taneous plaques (Stephen 1986).
In practice, diagnosis of dourine is based on clinical evidence
Keywords Diagnosis . Dourine . Identification . Pathological supported by serology (Office International des Epizooties (OIE)
lesions . Trypanosoma equiperdum 2008). Despite available serological tests are more sensitive, they
fail to distinguish between an active infection and a cured one
(Clausen et al. 1999). Detection of T. equiperdum, by standard
parasitological techniques, is usually difficult even in dourine
Introduction
positive horses, due to low numbers of parasites in the blood
or tissue fluids and chronic nature of the disease (Vulpiani et al.
Dourine is a contagious disease of equids caused by the pro-
2013). Additionally, diagnosis of the disease becomes more
tozoan parasite Trypanosoma equiperdum. Once widespread,
complicated in an area where the causative agents of surra or
dourine has been eradicated from many countries but is still
nagana occur and appears difficult to identify which parasite is
seen in horses in Asia, Africa, South America, Southern and
causing dourine (Hagos et al. 2010b).
Eastern Europe, Mexico, and Russia and was reported in
Dourine is a disease of great economic importance and well
documented as a trade barrier for the movement of horses
* Mulisa Megersa (Chin et al. 2013). Dourine poses a significant challenge to
mulisam38@gmail.com equine production, as transmission does not require insect
vectors that are influenced by climatic factors, the disease
1
College of Veterinary Medicine, Jigjiga University, P. O. Box: 1020, can be found anywhere and even the disease is more important
Jigjiga, Ethiopia in areas where mechanically or tsetse-transmitted
 Trop Anim Health Prod

trypanosomes are endemic. Though dourine still occurs in based on the morphology and molecular data from a study
many parts of the world, since its eradication from North on T. brucei, T. evansi, and T. equiperdum, other researchers
America and Northern Europe, research on the disease has suggested that T. evansi stocks distributed around the world
been neglected. The absence of published information on were derived from a mutated clone population of
many aspects of dourine should prompt experts in the field T. equiperdum that lacked maxicircle kDNA (Lun and
to bridge the gap in current knowledge about the disease pos- Desser 1995; Brun et al. 1998). To date, phylogenetic analyses
sibly through research or systematic review of existing litera- show that T. equiperdum and T. evansi are not monophyletic
ture. This paper presents a thorough review of the epidemiol- and should therefore be considered as subspecies of T. brucei,
ogy, diagnosis, and pathology of dourine. a parasite causing sleeping sickness in humans and nagana in
animals (Hébert et al. 2017). The possible phylogenetic rela-
tionship is illustrated in Fig. 1.
Equine Trypanosomosis (dourine) Unlike T. brucei whose kDNA contains hundreds of com-
plex heterogeneous minicircle sequence, T. equiperdum and
Definition and synonyms T. evansi share the same properties of minicircles which are
largely homogeneous and totally different from that of
Dourine is a chronic or acute contagious disease of equids T. brucei having only a single major minicircle class
transmitted directly from animal to animal during coitus (Gibson 2007; Lai et al. 2008; Lun et al. 2010). This strongly
(Calistri et al. 2013). The venereal disease of equines or supports the hypothesis that T. evansi is likely to directly arise
dourine has been also known under different other names from a mutated T. equiperdum which has a lack of maxicircles.
(Arabic Bel Dourin,^ English Bcovering disease,^ German Based on the biological and molecular evidence, it is sug-
BBeschalseuche,^ French BMal de coit,^ Russian BSlucnaja gested that T. equiperdum evolved from an ancient strain of
Boleznj,^ or BPodsedal^) (Hoare 1972). T. brucei which adapted to equine hosts, and that during the
period of adaptation, some parts of the maxicircle kDNA and
Etiology the heterogeneous minicircles were lost, causing the lack of de-
velopment within an insect vector (Lai et al. 2008). This is sup-
T. equiperdum is the causative agent of dourine that belongs to ported by the deletion of maxicircle sequences observed in at
the subgenus Trypanozoon (Hébert et al. 2017). This subgenus least two stocks of T. equiperdum (Lun et al. 1992). Lun et al.
also includes the three subspecies of T. brucei (Trypanosoma (1992) showed that Chinese T. equiperdum maxicircles are only
brucei brucei, Trypanosoma brucei gambiense, and about half the size of those of T. brucei, being approximately
Trypanosoma brucei rhodesiense), and T. evansi. T. b. brucei 14.3 kb in size. Because of the lack of parts of the maxicircle
causing nagana in domestic animals and T. b. rhodesiense and kDNA, cyclic stages no longer occurred in this mutated trypano-
T. b. gambiense causing sleeping sickness in humans. Further, some (Borst et al. 1987) and then this ancestral T. brucei isolate,
T. evansi causes surra predominantly in livestock but also in later called T. equiperdum, was finally limited to the equine hosts.
other mammals (Maudlin et al. 2004). Although great progress has been made in clarifying the
genetic and evolutionary relationships, many interesting ques-
Origin and identification of the parasite tions still need to be resolved with more evidence. In addition,
it is not clearly understood how or how often maxicircle
T. equiperdum is classified under the subgenus Trypanozoon kDNA loss has happened in dyskinetoplastic trypanosomes
along with T. brucei spp. and T. evansi; however, the species although it is clear that this phenomenon is frequently ob-
classification of Trypanozoon remains a controversial topic served in T. evansi and T. equiperdum both in vivo and
because it has been hypothesized that a very close evolution- in vitro. At the same time, the links between mechanical trans-
ary relationship exists among the trypanosome species of mission, the loss of kDNA, and host specificity remain uncer-
Trypanozoon (Suganuma et al. 2016). Based on biological tain (Wei et al. 2011).
and morphological characteristics, Hoare (1972) suggested It is difficult to distinguish T. equiperdum microscopically
that T. evansi evolved from T. brucei by adaptation to mechan- from other members of the subgenus Trypanozoon (T. evansi
ical transmission from host to host through their insect vec- and T. brucei). In particular, T. equiperdum and T. evansi can-
tors, while T. equiperdum was derived from T. evansi by not be differentiated on the basis of morphological criteria
adapting to the equine hosts. However, since T. evansi lacks (Claes et al. 2005). Like T. evansi, T. equiperdum is usually
kinetoplast DNA (kDNA) maxicircles (Lun et al. 1992), monomorphic. However, it sometimes exhibits pleomorphism
Hoare’s hypothesis that T. equiperdum arose from T. evansi like T. evansi during subpassages in rodents (Wei et al. 2011).
is inappropriate. In fact, there is no evidence to indicate that At the fine structural level, there are relatively more coated
the ability to reacquire kDNA occurred in trypanosomes or vesicles in the flagellar pocket of T. equiperdum, compared
other kinetoplastids (Lun et al. 1992). On the other hand, with that of T. evansi. It becomes somewhat difficult to
Trop Anim Health Prod

Fig. 1 Phylogenetic relationship

among three closely related
trypanosomes which indicates the
close relationship between
T. evansi and T. equiperdum
(Brun et al. 1998)

differentiate these two species with respect to the ultrastruc- always transmitted by an infected animal during copulation
tural properties (Brun et al. 1998). (OIE 2013). Horses usually die from infection without treat-
Neither parasitological nor serological tests are sensitive ment, whereas donkeys and mules are more resistant than
and specific enough, thus leading to various kinds of genetic horses and may remain unapparent carriers. Zebras have been
and molecular methods which have been continually updated tested positive by serology, but there is no conclusive evi-
in order to enhance greater precision in diagnosis of dence of infection (Brun et al. 1998). Since T. equiperdum is
Trypanozoon species and differentiation of these pathogens a tissue parasite found in equines in nature, its establishment in
(Wei et al. 2011). Accordingly, restriction fragment length the blood of laboratory animals is extremely difficult.
polymorphisms (RFLPs) (Lun et al. 2004), genome finger- However, once a strain becomes adapted to rodents, the para-
printing (Waitumbi and Murphy, 1993), and repetitive DNA sites can be maintained by serial passages, in the same manner
probes were used (Zhang and Baltz 1994). A series of tech- as T. evansi. It is noted that murine-adapted clones of
niques based on PCR have also been used, for example, mini T. equiperdum can cause acute infection like T. evansi when
satellite DNA analysis (Macleod et al. 2001), amplified frag- passaged through mice, rats, rabbits, horses, and dogs.
ment length polymorphism (AFLP) (Agbo et al. 2002), Domestic animals such as sheeps and goats infected with
multiplex-endonuclease genotyping (MEGA) (Claes et al. murine-adapted strain of T. equiperdum produce the clinical
2003), mobile genetic elements (MGE)-PCR, simple se- manifestations of dourine (Wang 1988).
quence repeat (SSR)-PCR (Li et al. 2005), and random ampli- Dourine has a worldwide distribution but few cases have
fication of polymorphic DNA (RAPD) (Lun et al. 2004). PCR been reported during the last three decades owing to the wide
test based on the RoTat1.2 variable surface glycoprotein use of artificial fertilization technology (OIE 2013). It was
(VSG) cDNA sequence was performed by Claes et al. once widespread during the times when the horse was mili-
(2004). Moreover, two kinds of techniques have been devel- tarily, economically, and agriculturally important. It was of
oped for detection and identification of African trypanosomes, great concern in the USA and Canada at the beginning of
i.e., fluorescence in situ hybridization with peptide nucleic the twentieth century. Nowadays, Western Europe, Australia,
acid probes (Radwanska et al. 2002) and the loop-mediated and the USA are considered to be free from dourine (Claes
isothermal amplification (LAMP) reaction (Thekisoe et al. et al. 2003). The infection is endemic in many areas of Asia,
2007; Njiru et al. 2008). However, despite the development Africa, Russia, Middle East, and Eastern Europe (OIE 2008).
of these, genetic and molecular techniques by different The latest official reports of dourine (i.e., Complement
scholars to clear species-specific identification within the sub- Fixation Test (CFT) positive cases) were in China,
genus trypanosome remains difficult. So far, the discovery of a Kazakhstan, Pakistan, Ethiopia, Botswana, Namibia, South
simple and reliable way to entirely distinguish all Africa, Brazil, Italy, and Germany (Fig. 2). However, due to
Trypanozoon species remains a big challenge. possible cross reactions in the CFT, it is difficult to conclude
that seropositive animals are real T. equiperdum cases
(Zablotskij et al. 2003). The prevalence of the disease in some
Epidemiology
countries is summarized in Table 1.
Host range and geographical distribution
Transmission
T. equiperdum has been reported to infect horses, donkeys,
and mules. There is no known natural reservoir of the parasite Unlike other trypanosomal infections, dourine is transmitted
other than infected equids (Brun et al. 1998). Infection is not almost exclusively during coitus. Dourine is the only
 Trop Anim Health Prod

Fig. 2 Distribution of dourine in

different parts of the world (Yonas
2015)

trypanosomosis that is not transmitted by an invertebrate vec- study conducted using clinical findings and laboratory and ep-
tor. T. equiperdum differs from other trypanosomes in that it is idemiological analyses of the outbreaks in Italy, based on fea-
primarily a tissue parasite that is rarely detected in the blood tures such as prevalence, age, reproductive activity, and rela-
(OIE 2013). tionship between the affected animals, indicated that the infec-
The trypanosomes, which are present in the seminal fluid tion is transmitted directly from animal to animal during coitus
and mucous membranes of the genitalia of the infected donor (Calistri et al. 2013). As the disease progresses, trypanosomes
animal, are transferred to the recipient during sexual inter- periodically disappear from the urethra or vagina; during these
course. Trypanosomes are rarely observed in the bloodstream periods, the animals are non-infective. Non-infective periods
of the host because they are normally localized in the capil- may last for weeks or months and are more likely to occur in
laries of the mucous membranes of the urogenital tract. the later stages of the disease. Thus, transmission is most likely
However, a few trypanosomes occasionally appear in the pe- in the early disease process (Wang 1988). An interesting find-
ripheral blood of animals with chronic infection. This could ing in the literature was a positive PCR test result from a pre-
provide the opportunity for bloodsucking insects to mechani- puce swab taken from a dourine-free stallion immediately after
cally transmit this parasite, although this is considered to be mounting an infected mare. The horse remained negative at all
very rare (Wang 1988). subsequent tests, supporting the theory that the parasite is pres-
The infection is more commonly transmitted from stallion to ent in the genital tissues but that sexual transmission is not
mare, facilitated by the presence of the parasite in the seminal constant (Vulpiani et al. 2013).
fluid and mucous exudates of the penis and its sheath. From the T. equiperdum can pass through intact mucous membranes
infected mare, the infection is transmitted to a stallion due to the and it is possible for foals to acquire infection by contamina-
presence of the parasite in the vaginal mucus (OIE 2013). A tion of nasal or conjunctival membranes with the vaginal

Table 1 Prevalence of dourine in

horses in some countries based on Countries Test employed Prevalence (%) Source
different tests
Botswana CFT 9.0 (Masupu and Majok 1998)
Ethiopia ELISA 19.26 (Hagos et al. 2010a)
Ethiopia Woo test, ROTat 1.2, and 18S PCR 4.6, 36.7, and 47.6 (Fikru et al. 2010)
Kazakhstan CFT 16.4 (Claes et al. 2005)
Mongolia CFT and ELISA 7.6 and 6.7 (Clausen et al. 2003)
Namibia CFT 8.33 (Kumba et al. 2002)
Italy CFT 0.54 (Calistri et al. 2013)
Trop Anim Health Prod

discharge. These infected foals can spread the organism when abdomen. In stallions of heavy breeds, the edema may extend
they mature. Other means of transmission may also be possi- over the whole floor of the abdomen (OIE 2013).
ble, but there is no evidence that arthropod vectors play any An observation made by Vulpiani et al. (2013) indicates
role in transmission. Intravenous or intraperitoneal experi- that infected stallions revealed mild signs than the infected
mental infections suggest that mechanical transmission by mares. Six months after infection, the stallions were almost
bloodsucking flies cannot be excluded. Foals born to mares asymptomatic. However, the differences with respect to sex
infected with T. equiperdum may be infected in utero or may cannot be statistically examined because of the low number of
become infected during parturition. Transmission to foals by considered cases in the study. An observation made by Watson
ingestion of infected colostrum or milk is considered rare (1920) indicates that apart from the fact of increasing viru-
(William and Steven 2007). The presence of trypanosomes lence resulting from continued passages accords with general
in the mammary secretions may support that the infection experience that the disease is usually more progressive in the
can occasionally pass to foals during suckling (Pascucci stallion than in the mare.
et al. 2013). Foals that ingest colostrum from infected mares
will become seropositive due to passive transfer of antibodies; Pathological lesions of dourine
these foals are usually seronegative by from 4 to 7 months of
age (William and Steven 2007). The disease is characterized by edematous lesions of the gen-
italia, involvement of the nervous system, and progressive
Clinical signs emaciation, and it is ultimately fatal in most cases. Typical
cutaneous lesions, from which the disease derives its name
The incubation period between exposure and initial clinical Bdourine,^ have been described as circular elevated plaques
signs is highly variable; it may be as short as 1–2 weeks or of thickened skin ranging in size from 1 to 10 cm in diameter,
as long as several years (William and Steven 2007). Clinical resembling money or Bdouros^ (Claes et al. 2005). The con-
signs of dourine are highly variable in manifestation and se- stant antigenic variations of the parasite result in the release of
verity. The disease is characterized mainly by swelling of the a large amount of biological active products and the formation
genitalia, cutaneous plaques, and neurological signs but sever- of immune complexes, which are certainly major factors in
ity varies with the virulence of the strain, the nutritional status triggering a variety of clinical and pathological changes
of the horse, and stress factors. Clinical signs often develop (Zwart 1989).
over weeks or months, frequently waxing and waning with
relapses, probably precipitated by stress. This can occur sev- Gross pathological lesion
eral times before the animal either dies or experiences an ap-
parent recovery. The mortality rate is believed to be in excess Dourine is characterized by cachexia, anemia, muscular
of 50% (Sidney et al. 2013). hypotrophy, ataxia, and lack of coordination of the hindquar-
A number of authors have broken the course down into ters, ptosis of the lower lip, genital lesions, skin edematous
three stages: stage 1 (genital lesions), stage 2 (cutaneous plaques, and peripheral edema (Pascucci et al. 2013). The
signs), and stage 3 (nervous signs). Stage 1 involves genital presence of nervous signs without sensory alterations seems
edema and swelling, manifesting 1–2 weeks after infection. In to confirm the tropism of T. equiperdum for the peripheral
stage 2, typical cutaneous plaques (Bsilver dollar^ plaques) rather than the central nervous system, in contrast with other
appear, with thickening of the skin, considered pathognomon- trypanosomes (Berlin et al. 2009).
ic by some authors. Stage 3 is characterized by progressive At postmortem examination, gelatinous exudates are pres-
anemia, neurological disorders, and paresis of the hindquar- ent under the skin. In the stallion, the scrotum, sheath, and
ters, often ending in death (Claes et al. 2005). testicular tunica are thickened and infiltrated. In some cases,
A pathognomonic sign is the edematous plaque consisting the testes are embedded in a tough mass of sclerotic tissue and
of an elevated lesion in the skin, up to 5–8 cm in diameter and may be unrecognizable. In the mare, the vulva, vaginal muco-
1 cm thick. The plaques usually appear over the ribs, although sa, uterus, bladder, and mammary glands may be thickened
they may occur anywhere on the body, and usually persist for with gelatinous infiltration. The lymph nodes, particularly in
between 3 and 7 days. They are not a constant feature. Pyrexia the abdominal cavity, are hypertrophied, softened, and, in
is intermittent; nervous signs include incoordination, mainly some cases, hemorrhagic. The spinal cord of animals with
of the hind limbs, lips, nostrils, ears, and throat. paraplegia is often soft, pulpy, and discolored, particularly in
Depigmentation of the genital area, perineum, and udder the lumbar and sacral regions (OIE 2013).
may occur. In the stallion, the first clinical sign is a variable The presence of dourine infection in the stallions did not
swelling involving the glans penis and prepuce. The edema appear to interfere with libido or the ability to achieve erection
extends posteriorly to the scrotum, inguinal lymph nodes, and even where there is pronounced edema of the scrotum and
perineum, with an anterior extension along the inferior sheath. Similarly, the presence of infection did not appear to
 Trop Anim Health Prod

adversely affect the fertility of either stallions or mares. This Diagnosis

study also reported on five occasions clean mares conceived to
services by infected stallions and on three occasions infected Diagnosis of dourine is a challenge, due to limited knowl-
mares conceived to services by clean stallions. Two foals born edge about the parasite and host-parasite interaction fol-
to infected mares were normal and were reared to maturity lowing infection. In practice, diagnosis is based on clinical
(Barrowman 1976). evidence supported by serology (Alemu et al. 1997; Hagos
et al. 2010a). Clinical signs of dourine can provide a strong
Microscopic lesions indication of the presence of the disease, but confirmatory
diagnosis is needed (Claes et al. 2005). The incubation
On histological examination of tissue samples, the disease is period may vary from a few weeks to several years, and
characterized by hemosiderin deposition in the spleen, the some of the clinical signs, which include genital edema,
iliac, supramammary, and popliteal lymph nodes showed weight loss, skin lesions known as silver dollar plaques,
non-specific reactivity with hyperplasia of the plasma cells, and neurological signs, may be absent in the early stages or
a sign of increased hemolymphatic activity. The edematous during latent infections (Luckins et al. 2004; Claes et al.
plaque showed a characteristic picture of pustular dermatitis, 2005). Diagnosis of dourine, therefore, requires confirma-
particularly severe around the lesion, with severe inflamma- tion by parasitological, serological, and molecular
tion and vacuolar degeneration extending to the deepest layers techniques.
of the skin, with involvement of the cutaneous adnexa and
perivascular plasma cell inflammation. There was exudates Parasitological diagnosis
of cell detritus in the same area, mainly eosinophils and the
bodies of free parasitic protozoa, in a picture described as Wet and thick blood films In this test, 5–10 μl of blood is
Btrypanosomal sand^ (Pascucci et al. 2013; Scacchia et al. placed on a slide and examined microscopically at ×400 mag-
2013). nification under a cover slip. Trypanosomes are observed
In the nervous system of infected horses, neurodegener- moving between the erythrocytes in infected animals. It has
ative lesions and inflammatory vasculitis of the central very low sensitivity, with a detection limit as high as
nervous system with edematous infiltration in the facial 10,000 trypanosomes/ml, but it is still in use because of its
and lingual nerves were reported. In the udders, there are low cost and simplicity. Giemsa or Field-stained thin blood
histological lesions attributable to severe interstitial in- films have a similarly low sensitivity. It is time consuming
flammation accompanied by strong supramammary lymph (10–20 min per slide) and requires expertise to recognize the
node reactivity and the presence of Russell’s bodies. Liver parasite (Murray et al. 1979).
showed multifocal areas of hepatitis while the kidneys are
affected by plasma cell inflammation of the renal pelvis. Microhematocrit centrifugation technique Microhematocrit
Periglandular inflammation in the vulva, vagina, uterus, centrifugation technique (mHCT), a blood concentration tech-
and clitoris was also observed in the infected horse. The nique (also called the capillary tube centrifugation technique
constant finding of iliac and supramammary lymph node or the Woo test), is the most frequently applied concentration
positivity and lymphatic activity on both macroscopic ob- technique with better sensitivity than direct microscopic ex-
servation and histological examination seem to confirm amination. In this test, capillary tubes containing anticoagu-
that the parasite spreads mainly through the lymphatic sys- lants are filled three-quarters full with blood. The dry end is
tem (Pascucci et al. 2013). sealed with plasticine. By high-speed blood centrifugation in a
Although, depigmentation around the perineum is often hematocrit centrifuge for 6–8 min, trypanosomes are concen-
described as characteristic of clinical cases of dourine trated between the red blood cells and the plasma, together
(Claes et al. 2003; Hagos et al. 2010a; Vulpiani et al. with the white blood cells. The capillary tubes mounted in a
2013), no microscopic description of such lesions was cit- special viewing holder can be directly examined at low mag-
ed in previous literatures. Severe dermatitis with hydropic nification (×10 or ×40) for motile parasites. The estimated
degeneration and necrosis of the keratinocytes of stratum detection threshold of mHCT is 500 trypanosomes/ml of
spinosum and necrosis of basal cells including the melano- blood sample (Reid et al. 2001).
cytes with excess free melanin pigment within the epider-
mis were reported recently. The probable cause of depig- Mini anion-exchange centrifugation technique The mini
mentation around the vulval skin of infected mares could anion-exchange centrifugation technique (mAECT) con-
be due to severe necrosis of melanocytes, as the sists of separating the trypanosomes which are less nega-
depigmented areas were microscopically characterized by tively charged than blood cellular components from ve-
severe necrosis of cells, excess free melanin, and formation nous blood via anion-exchange chromatography and final-
of cystic structures in the epidermis (Yonas 2015) (Fig. 3). ly concentrating them at the bottom of a plastic collector
Trop Anim Health Prod

Fig. 3 a Depigmentation of the

vulval lip (gross). b Vacuolar
degeneration of the cells (lighter
arrows) and necrotized cell
(darker arrow) in the stratum
spinosum, degeneration and
necrosis of the basal cells with
melanin pigment were evident
(circled areas). c Excess free
melanin in the stratum spinosum
(small circles) and within the
basal layer (large circles). d
Severe dermatitis with infiltration
of lymphocytes and plasma cells A B
in the epidermis and dermis
(circled areas) (Yonas 2015)

C D

tube by low-speed centrifugation. The tip of the glass tube Serological techniques
is then examined in a special holder under the microscope
for the presence of trypanosomes. The large blood volume It is extremely difficult to detect the parasite in the body
of up to 300 μl enables the detection of less than 100 try- fluids of infected horses (Claes et al. 2005); therefore, di-
panosomes/ml, resulting in high sensitivity. However, the agnosis of T. equiperdum by standard parasitological tech-
manipulations are quite tedious and time consuming (Reid niques is difficult, owing to the low numbers of parasites in
et al. 2001; Buscher et al. 2009). the blood or tissue fluids. Consequently, the demonstration
of trypanosomal antibodies in the serum has become the
most important parameter determining the disease status of
Animal inoculation Repeated attempts have been made by individual animals (Bishop et al. 1995). Trypanozoon
different workers (Alemu et al. 1997; Clausen et al. 1999, group-specific trypanosomal antigen could be of use in
2003) to demonstrate and isolate T. equiperdum in labora- an antibody assay for the diagnosis of T. equiperdum in-
tory mice but all were unsuccessful. However, once a strain fections. However, based on anecdotal evidence, it appears
becomes adapted to rodents, the parasites can be main- that T. equiperdum-infected laboratory animals and horses
tained by serial passages, in the same manner as T. evansi suspected of dourine also positively react in the Card
(Luckins 1994). Under laboratory conditions, dogs can be Agglutination test trypanosomiasis (CATT)/T. evansi and
infected with T. equiperdum as reported by Rouget (1986). Enzyme- linked Immunosorbent Assay (ELISA)/T. evansi
In experimental infections carried out in the Institute for prepared with fixed whole trypanosomes of the RoTat 1.2
Tropical Medicine to raise antisera against VSGs, rabbits VAT (Claes et al. 2003).
infected with the available laboratory strains developed CATT/T. evansi test is fast, uses a standardized antigen, and
clinical signs that could not be distinguished from those can be performed in situ, i.e., without the need of a fully
developed by rabbits infected with T. evansi (Verloo et al. equipped laboratory. Recently, it has been proven that most
2001). Owing to the marked predilection of T. equiperdum so-called T. equiperdum strains also express isoVATs of
for the testicles of rabbits, some authors recommended T. evansi RoTat 1.2. Therefore, the CATT/T. evansi may prove
intratesticular inoculation of these animals for the diagno- to be a good test for equine trypanosomosis, regardless wheth-
sis of dourine in equines. Ruminants were refractory to er the causative agent is T. evansi (surra) or T. equiperdum
infection with T. equiperdum (Hoare 1972). (dourine) (Claes et al. 2003).
 Trop Anim Health Prod

The complement fixation test is the most commonly Molecular techniques

used OIE-prescribed serodiagnostic test developed for
T. equiperdum and successfully used as part of a program Although no T. equiperdum-specific polymerase chain reac-
to eliminate T. equiperdum from North America. It is still tion (PCR) method is available, subgenus Trypanozoon-spe-
used for international trade in monitoring horses for ex- cific PCR can be used for detection of T. equiperdum DNA.
port/import. Despite the usefulness and universal accep- Recently, a highly sensitive real-time PCR for Trypanozoon
tance of the CFT for diagnosing dourine, some discrepan- subgenus was applied on tissues and fluid samples from a
cies have been recorded. The disadvantages of the CFT are naturally dourine-infected horse, enabling the detection of
that it requires careful continuous titration of numerous low numbers of the parasites (Scacchia et al. 2011; Pascucci
labile agents and that it does not function with sera having et al. 2013). PCR and other related DNA amplification
anticomplementary activity. CFT is not species specific, methods have been used to examine exudates or tissue sam-
but only specific for the subgenus Trypanozoon. The draw- ples, taking into account their failure on blood samples after
back of the test is lower specificity where it cannot differ- the initial phase of the infection (Calistri et al. 2013).
entiate T. equiperdum from other similar trypanosomes. Direct diagnosis based on molecular techniques can be
Hence, the diagnostic significance of CFT is therefore highly sensitive for parasite detection in body fluids such as
doubtful in countries where both T. equiperdum and the blood (Becker et al. 2004). However, this approach is
T. evansi infections occur in equines (Luckins 1994). difficult to apply for mass screening and negative results do
Although the CFT has been in use for many years for not exclude the possibility of infection. In fact, T. equiperdum
diagnosis of dourine, it is considered to be less sensitive multiplies predominantly in extracellular tissue spaces and is
than ELISA and IFAT for the detection of the serum anti- seldom found in peripheral blood (Theis and Bolton 1980).
bodies against T. equiperdum (Wassal et al. 1991; Bishop Diagnosis of T. equiperdum infection is thus still strongly
et al. 1995). based on serological evidence.
Indirect fluorescent antibody test is frequently used for the
diagnosis of dourine, as a confirmatory test for CFT results, Treatment
since immunofluorescence provides a reliable and sensitive
technique. But its interpretation is both subjective and labor Pharmaceutical therapy is not recommended because animals
intensive, and it is therefore more suited to the testing of small may improve clinically but remain carriers of the parasite
numbers of sera (Williamson et al. 1988). (OIE 2013). There are no officially approved drugs to treat
The use of ELISA for routine diagnosis of dourine horses suffering from dourine although some older publica-
would provide a significant advantage over current sero- tions mentioned experimental treatment of horses with
logical tests if a defined antigen was used, since it would suramin and neoarsphenamine (Ciuca 1933) or
permit test standardization and more readily allow compar- quinapyramine sulfate (Vaysse and Zottner 1950). Evidence
ison of tests among laboratories. It additionally, lends itself from in vitro drug sensitivity determination of T. equiperdum
to a considerable degree of automation, which makes it indicates that suramin, diminazene, quinapyramine, and
suitable for a large number of samples (Wassal et al. cymelarsan are effective (Zhang et al. 1992; Brun and Lun
1991). Different workers have stated that the ELISA has 1994). These were also supported by other researchers who
a satisfactory concordance ratio with CFT and can be used found cymelarsan® quite effective in curing horses at both
to supplement CFT (Williamson et al. 1988; Alemu et al. 0.25 and 0.5 mg/kg in acute as well as chronic form of dourine
1997). There are also several other alternative serological (Hagos et al. 2010b). Brun and Lun (1994) reported drug
tests that are used, such as the agar gel immunodiffusion sensitivity of T.equiperdum isolates in vitro and found the
test, the arrayed immunodiffusion method (Hagebock et al. isolate was highly sensitive to melarsorol, isometamidium,
1993), and the competitive immunoassay (cELISA). The and suramin; with regard to diminazene, T.equiperdum was
cELISA method has several advantages over the CFT: it not sensitive as the most sensitive T. evansi strains.
can be performed in less time than the corresponding CFT
procedure, it is reproducible, results are objectively mea- Prevention and control
sured and calculated, and the method is amenable to auto-
mation (Katz et al. 1999). While serological tests can be There is no vaccine available for dourine. As dourine is pri-
the method of choice for mass screening of populations, marily a venereal disease, prevention of natural mating or AI
their main limitation will remain as the failure to demon- with infected horses (stallions or mares) or infected stallion
strate the parasite. Unfortunately, parasitological tech- semen is the most important means of control. Prevention of
niques are known to lack sensitivity, especially for the de- dourine is therefore based on the establishment of freedom
tection of T. equiperdum, which is considered to be a tissue from infection, and this is done by testing blood for the pres-
parasite rather than a blood parasite (Brun et al. 1998). ence of antibodies against T. equiperdum, which is more
Trop Anim Health Prod

reliable than testing for the presence of the protozoan parasite Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
itself. Any introductions of horses from endemic areas or areas
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
of incursion should be isolated and the blood tested for anti- distribution, and reproduction in any medium, provided you give appro-
bodies by complement fixation test (Sidney et al. 2013). priate credit to the original author(s) and the source, provide a link to the
Control of the disease depends on compulsory notification, Creative Commons license, and indicate if changes were made.
slaughter of infected animals, and movement control enforced
by legislation in most countries (OIE 2013). Dourine should
be eradicated from an incursion into a non-endemic area by References
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