Haemoparasites—Challenging and Wasting Infections in Small Ruminants: A Review

Simple Summary Vector-borne haemoparasites in small ruminants are widespread, although only scattered information is available on their occurrence. Haemoparasites occur in the genera Anaplasma, Babesia, Ehrlichia, Mycoplasma, Theileria and Trypanosoma, of which several species cause challenging and wasting conditions with severe impact on the small ruminant industry. However, the controls of these infections are challenging tasks, especially since several of the available medical drugs have negative environmental consequences. In addition, vectors may spread across the world to new geographical areas, especially related to climate change and increased globalization. These changes may have a profound impact on infection ecology and disease management. Integrated control strategies should be implemented, such as breed resistance against vectors and infections, and strategic vector and infection control. Abstract Haemoparasites include bacteria, mycoplasma, protozoa and flagellates inhabiting the bloodstream of living hosts. These infections occur worldwide and are transmitted by vectors, especially ticks and tsetse flies. Geographical distribution varies due to movements of animals and vectors between geographical areas, and even between countries and continents. These changes may be caused by climate change, directly and indirectly, and have a huge effect on the epidemiology of these microbes. Active and ongoing surveillance is necessary to obtain reliable maps concerning the distribution of these infections in order to do proper risk assessment and efficient prophylactic treatment. Genera Anaplasma, Ehrlichia, Mycoplasma, Babesia, Theileria and Trypanosoma include common haemoparasite species in small ruminants causing a variety of clinical manifestations from high fatality rates to more subclinical infections, depending on the species or strain involved. These infections may also cause ill-thift or long-lasting wasting conditions. Life-long infections are a common feature of these pathogens. The present review will focus on haemoparasites in small ruminants, especially related to challenging and wasting infections.


Introduction
In recent years, a series of vector-borne diseases have spread to new geographical areas across the world, of which several are caused by haemoparasites. Haemoparasites are pathogens that inhabit the bloodstream of the host and includes microorganisms such as bacteria, mycoplasma, protozoa and flagellates. These infections occur on all continents, whereas the distribution of these infections changes continuously due to the migration and transportation of vectors and animals and an increased globalization of both live animals and their products. Short-and long-distance movements of vectors and hosts are driven directly or indirectly by climatic changes. These changes will have a huge effect on the distribution and establishment of both pathogens and vectors [1,2]. Climate change may provide new habitats for living organisms and cause a climatic-driven behaviour adaptation that may ease

Species and Distribution
Mycoplasma ovis (formerly classified as Eperythrozoon ovis) is an uncultivated pleiotropic bacterium which parasitizes erythrocytes [11]. In the last decade, a similar organism, Candidatus Mycoplasma hemovis, has been detected. However, recent investigation suggests that these two species may represent the same organism [12].
M. ovis occurs worldwide in small ruminants, although there is a lack of information concerning the actual distribution, especially in goats [11,13].

Transmission and Reservoir
Transmission of M. ovis seems to occur mainly by biting insects (flies, lice, keds and mosquitos), but also by needles or surgical instruments. Ticks may occasionally play a role in pathogen transmission [11]. In addition, oral transmission has been shown to occur-whereas only one infected erythrocyte seems to be enough to transmit the infection [14]. No indication of vertical transmission has so far been reported.
Natural reservoirs besides sheep and goats are unknown; however, cervids seem to be infected with M. ovis [15,16]. In recent years, human infections have been reported [17,18].

Clinics and Diagnosis
Mycoplasma ovis causes haemolytic anaemia in small ruminants. Clinical symptoms also include hemoglobinuria, unthriftiness, stiffness and reduced weight gain [14,19]. Fever and icterus are not regularly observed. However, subclinical or latent infections seems to be the rule. Clinical disease is often triggered by other factors such as malnutrition or gastrointestinal parasitism. Susceptibility to the infection seems to vary between sheep and goats [20], of which clinical disease is rarely observed in goats [13]. In sheep, however, chronic infection may occur, an ill-thrift condition in lambs where they become small, pot-bellied, emaciated and often with a fatal outcome [14,21]. M. ovis may lead to severe livestock losses mainly due to retardment of weight gain in lambs and loss of condition in mature sheep [22].
The organism can be found on erythrocytes by light microscopy with the use of stained blood smears [23]. Up to 100% of the erythrocytes can be infected, although normally only a few percentages are detected by microscopy. Low infection level may be difficult to diagnose by direct microscopy and require examination of repeated blood smears. Serology can be used however molecular methods are often necessary in order to verify the diagnosis [11]. Co-infections with other pathogens (such as Anaplasma ovis and A. phagocytophilum) may occur, aggravating the clinics and challenging the diagnostics [17].

Treatment and Control
Neoarsphenamine, antimosan or oxytetracycline have been recommended for sheep. No specific treatment evaluations have been reported in goats [13]. Treatment may diminish clinical symptoms but may not clear the organisms from the body. Controls involve good preventive measures such as enough nourishment of good quality, minimizing the risk of biting flies and avoiding the spread of the organism by needles and surgical instruments.

Species and Distribution
Babesiosis is caused by protozoa in the genus Babesia, of which the main species affecting small ruminants are B. ovis, B. motasi and B. crassa. B. ovis is the most pathogen species and an important tick-borne haemoparasite in sheep worldwide [24], while B. motasi is the dominant species in goats [13]. B. motasi may consist of at least two species/subspecies, which may differ in pathogenicity [24,25]. The situation regarding Babesia spp. of small ruminants is, however, rather confusing and includes several new and unclassified strains/species [25,26].
B. ovis has a worldwide distribution, while B. motasi has been detected in Europe, central and northern Africa, Asia and Central America [24,27] B. crassa has only been detected in a few countries, including Iran and Turkey, and is known only in areas were Hyalomma and Rhipicephalus ticks are common [13].

Transmission and Reservoir
Rhipicephalus ticks, such as R. bursa, R. sanguineus and R. turanicus have been implemented in the transmission of B. ovis, whereas R. bursa is the only vector so far that is reported to transmit B. ovis transovarially [28]. Other tick species may occasionally be involved in the transmission cycle [29,30]. In contrast, B. motasi is mainly associated with Haemophysalis punctata or other ticks in the genus Haemophysalis [13]. Sheep and goats are the only known vertebrate reservoir hosts of B. ovis and B. motasi. In addition, ticks may serve as natural reservoirs [31]. B. ovis is known to be maintained in several generations of ticks without reinfections [32].

Clinics and Diagnosis
Acute babesiosis caused by B. ovis is characterized by apathy, fever, anaemia, jaundice and haemoglobinuria, and mortality may occur. The infection varies in the degree of severity associated with age, immunity and health status. Young animals seem to develop less clinical symptoms due to innate immunity. Infection is normally mild in indigenous sheep, whereas severe signs are often related to animals introduced from non-endemic areas. In goats, natural infection with B. ovis has only been reported in few areas and only as a subclinical condition [13,33]. Animals that survive the acute infection become low-level carriers and could be persistently infected for years without apparent clinical signs [34]. However, relapses may occur under stressful conditions, such as poor nutrition and other infections [31].
B. motasi produces in general a mild clinical response characterized by fever, haemoglobinuria and anaemia, but is rarely responsible for significant death losses [30]. However, more severe symptoms have been recorded in goats with high morbidity and mortality. Death may occur 48 h after the onset of clinical symptoms. Chronic and wasting condition may occur with anaemia and ill thrift [13]. In contrast, B. crassa infection in small ruminants seems to be non-pathogenic [34].
Diagnosis is based on the clinical symptoms mentioned above and the demonstration of typical protozoa in stained blood smears. However, the infection rate rarely exceeds a few percentages during the acute phase of the infection and may be difficult to detect by blood smear microscopy [30,31]. However, PCR and serological tests are available, especially PCR-analysis should be used to detect a subclinical B. ovis infection [27,28]. The most characteristic findings at post-mortem are anaemia, icterus and haemoglobinuria.

Treatment and Control
Earlier results indicate that treatment of babesiosis (B. ovis infection) with imidocarb diproprionate was efficient [35]. In addition, diminazene aceturate may be efficient against B. ovis The development of new drugs is in progress [36]. In goats, diminazene has been used against B. motasi [13]. Tick control is important in order to reduce the occurrence of babesiosis (see below).
Theileria species in small ruminants are widespread, particularly in tropical and subtropical regions of Africa, the Middle East, eastern and southern Europe and Asia [41], although only scattered information is available.

Transmission and Reservoir
A variety of tick vectors are involved, mainly in the genera Haemaphysalis, Hyalomma and Rhipicephalus [42] (Table 1). For instance, T. lestoquardi is mainly transmitted by Hyalomma anatolicum and Haemophysalis qinghaiensis, and T. ovis by R. bursa and H. punctata. Transplacental transmission of T. lestoquardi has been reported in both sheep and goats [43,44]. In addition, T. luwenshuni and T. uilenbergi seem to be related to H. qinghaiensis and H. longicornis [39]. Limited data are available concerning natural hosts of the different Theileria species. T. lestoquardi and T. ovis have so far only been reported from sheep and goats, while T. uilenbergi and T. luwenshuni have also been detected from sika and red deer [42].

Clinics and Diagnosis
Only T. lestoquardi, T. luwenshuni and T. uilenbergi are considered pathogenic for small ruminants, of which T. lestoquardi is the most pathogenic species [40,45]. Susceptibility to the infection varies due to internal (genetics, breed, lactation, parturition) and external (nutrition, concomitant infections) factors. In addition, the infection dose may influence the outcome of the infection, whether it will become subclinical or lethal [37].
T. lestoquardi, causing malign theileriosis, may introduce severe infection in sheep with high mortality rates (46-100%) [30,46]. In malign theileriosis, an acute form is common, but subacute and chronic forms may also occur. Clinical signs in the acute form are fever, cessation of rumination, swelling of superficial lymph nodes, diarrhoea, jaundice and haemorrhages (submucosal and subcutaneous). In chronic infections, intermittent fever, inappetence, anaemia, jaundice and emaciation are recorded. In general, Theileria spp. seems to cause much less symptoms in goats than in sheep [13].
Other Theileria species may cause mild or even inappreciable clinical symptoms, such as T. ovis causing benign theileriosis [24]. However, benign Theileria species may also cause significant production losses in imported, immunocompromised or stressed animals [37].
Diagnosis is based on the detection of protozoa in stained blood smears, lymph node biopsies (live animals) or in lymph nodes or spleen smears at post-mortem [30]. However, parasitaemia fluctuates and may drop below detection level, so microscopic detection of parasites can be difficult and does not readily allow for differentiation between species [47]. Serology are useful for identifying infected animals, although not specific to identify species. Detection of Theileria in the blood should be performed by molecular methods, such as reverse line blot and real-time PCR [48,49]. Post-mortem analysis includes anaemia, icterus, intramuscular and subcutaneous oedema, splenomegaly and hepatomegaly [37].

Treatment and Control
Specific treatment of caprine theileriosis is lacking. For T. lestoquardi, a single injection of parvaquone or buparvaquone given at two occasions may be used. A single dose of halofuginone is also reported to be efficient [18,30]. Theileria species causing mild or even inappreciable clinical symptoms is usually not treated. A vaccine is not commercially available. Tick control measures should be considered for controlling the disease and in order to limit the distribution and expansion of the pathogenic species. However, several of these treatments are not environmentally friendly and may also cause chemical residues in animal products. Control should be based on an integrated control against both parasites and vectors.

Species and Distribution
Trypanosomiasis is caused by haemoflagellates in the genus Trypanosoma. Several species are involved although not all of them seem to be pathogenic for small ruminants. Animals are often infected with more than one species or strain [50]. The most important species for small ruminants are T. congolense, T. vivax, T. brucei and T. evansi [51]. However, recent molecular methods indicate that T. evansi may is a subspecies or strain of T. brucei [52]. In addition, several strains of trypanosomes occur within each species [13].
As already mentioned, the most important species for small ruminants are T. congolense, T. vivax, T. brucei and T. evansi [10]. However, their real distribution is unknown. The infection is widespread in tropical and subtropical areas, especially in sub-Saharan Africa, but Trypanosoma species have also been detected in Europe, West Indies, Central and South America and Asia [13,30]. Outside Africa, T. evansi have been established in Asia, while both T. vivax and T. evansi have been established in South America [10,30].

Transmission and Reservoir
In Africa, most cases seem to be transmitted by tsetse flies (Glossina spp.), mainly in sub-Saharan Africa. However, the geographical distribution of trypanosomes extends beyond the tsetse-infested areas, which may be due to animal movement and mechanical transmission by other vectors. T. evansi and T. vivax have, for instance, been shown to be mechanically transmitted by blood-sucking insects (especially in the Stomoxydae and Tabanidae families) in South America and Asia [10,30]. However, cyclic transmission in the vector seems only to involve Glossina species. In addition, Trypanosma spp. may be transmitted by contaminated needles during medication and vaccination, which seems to be one reason for the widespread distribution in tsetse-free areas [53]. Transplacental transmission of T. evansi and T. vivax in sheep has also been reported [53,54].
Concerning the animal reservoir, little is known about the natural animal reservoir. Trypamosomes usually affect several animal species. Infected sheep and goats are carriers and can be the main source for maintenance of the infection [55]. Small ruminants may also function as a reservoir of trypanosomes affecting other mammalian species including humans [56].

Clinics and Diagnosis
The main clinical symptom related to trypanosomiasis is anaemia. Infected animals may show intermitting fever, decreased appetite, anorexia, rumen atony, enlarged lymph nodes and progressive loss of condition. Jaunice and hemoglobinuria are uncommon findings. Reduced milk production, reduced fertility and increased mortality may also be an indication of trypanosomiasis [10]. Clinical signs depend on the species of Trypanosoma involved. T. vivax and T. congolense cause acute, subacute and chronic disease, in which the acute form may cause death or recovery within four to six weeks. In chronic cases, severe emaciation may occur. T. congolense infection often results in cyclic fever and parasitaemia, and the mortality rate is high if the infection has lasted for more than 12 weeks. In T. brucei cases, CNS-symptoms such as head pressing, circling and opisthotonus may occur, often with a fatal outcome [13].
Goats with chronic trypanosomiasis may be more susceptible to helminthiasis probably as a result of immunosuppression. A reduction in milk quality and production have been observed in T. vivax-infected goats [57]. In addition, trypanosome infection in goats may be associated with ovarian dysfunction, irregular estrus cycles and testicular atrophy [13].
Variation of the clinical presentation is caused by several factors, involving strains/species of trypanosomes and genetic variation within and between breeds. It is important to notice that chronically infected animals can remain subclinical for a long period. However, reactivation of clinical symptoms may occur due to nutritional and physical stress or infection with other pathogens. Infection should therefore always be suspected in animals showing relapsing fever, progressive weight loss, abortion and especially anemia [55].
Anemia and emaciation in animals from areas with tsetse flies indicate trypanosomiasis. Diagnosis is based on organisms found in stained blood smears or tissues. However, trypanosomes may be difficult to identify in chronic infections due to low parasitaemia. To confirm T. brucei infection, examination of lymph node aspirate or mouse inoculation are the preferred methods. Several serological tests for antibody detection are available. In addition, PCR methods are developed, but demand well-equipped labs. Necropsy of acute cases will reveal anaemia, lymph node enlargement, splenomegaly, petechial bleedings (mucosa/serosa) and hydropericardium [13].

Treatment and Control
Trypanosomiasis has for decades been a major concern in small ruminant production, especially in sub-Saharan Africa [13]. In the African countries, it has been estimated that around 260 million small ruminants have reduced productivity due to these haemoparasites [32].
Over the years, extensive efforts have been made to control and eliminate the tsetse flies, based on the ground spraying of insecticide, sterile insect release technique, bush clearing and destruction of fly habitat, use of traps and the use of insecticide-treated livestock [10]. However, these interventions have only partly been successful and are often associated with negative environmental consequences such as destruction of the insect fauna, pollution of water bodies and deforestation. In addition, residues may occur in animal products [58]. Curative and prophylactic use of trypanocidal drugs administrated by the farmers, remains the most important method of controlling animal trypanosomiasis [9]. A limited number of trypanocidal compounds are available, such as diminazene aceturate, isometamidium and homidium chloride. However, the use of a few therapeutic drugs with a limited efficacy have fueled the widespread emergence of resistance [58]. In addition, there are numerous constraints on the control of trypanomosiasis, such as reservoirs in the wild animal populations, lack of vector control and lack of economic resources [13].
The most important method in the future to control this infection may be breeding for trypanotolerance, since indigenous breeds seem to tolerate the infection and perform better than imported breeds or crossbreeds. Trypanotolerance offers farmers an important option for sustainable production in risk areas. Preliminary field studies indicate that sheep and goat breeds such as Dwarf West African goat, Small East African goat, Djallonke sheep and Red Masai sheep are trypanotolerant. In these cases, trypanotolerance seems to be more related to resilience than to resistance [59]. However, factors such as helminthiasis, inadequate nutrition level and the severity of the trypanosome infection involved (strain, dose) may have a negative effect on this tolerance [13,51].
In sub-Saharan Africa, the major fronts against trypanosomiasis are the reduction or elimination of the tsetse populations. No effective vaccine is yet available, due to challenges identifying proper vaccine candidates [13]. The lack of a suitable vaccine has facilitated an overuse of the few available drugs. In addition, ongoing efforts to control the tsetse flies have been largely ineffectual, partly due to political instabilities and armed conflicts in the actual areas [58]. Risk-based control and monitoring activities are the foundation to control disease outbreak of trypanosomiasis [10].

Haemoparasites Rarely Involved in Wasting Disease
Haemoparasites in the genera Anaplasma and Ehrlichia are also important infections in the small ruminant industry, which seldom cause wasting conditions, except for tick pyaemia related to a primary A. phagocytophilum infection. The most common infections among these species will only briefly be mentioned.

Anaplasma ovis
A. ovis is a rickettsia with a widespread infection in small ruminants, especially in the Mediterranean countries and central Europe. A. ovis is spread by a variety of ticks, particularly Rhipicephalus and Dermacentor species. The infection in small ruminants may cause haemolytic anaemia, weight loss, abortion and even death [60]. However, subclinical infection is common, and an active surveillance is necessary in order to investigate the real distribution [61]. Outbreaks of severe illness in sheep and goats are rare and seem to occur mainly under stressful conditions. However, infection with A. ovis may predispose animals to other infections and parasite infestation resulting in clinical disease or even death and may therefore have an impact on health, milk and meat production in small ruminants [61].
Recently, outbreaks of anaplasmosis in lambs due to A. ovis, have been reported, including severe anaemia and icteric carcasses [62,63]. The pathogenesis of this strain has to be further unravelled.

Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila)
A. phagocytophilum causes tick-borne fever (TBF) in small ruminants [64]. The infection is mainly associated with Ixodes spp. ticks. A. phagocytophilum infection is widespread in Europe, while only scattered information is available from other continents. Only a few cases of A. phagocytophilum infection in goats have so far been reported.
TBF may cause severe economic and welfare challenges in the sheep industry. The most characteristic symptom of the disease in domestic ruminants is high fever. TBF is seldom fatal, unless complicated by other infections. The infection may cause immunosuppression in sheep that makes affected animals vulnerable to secondary infections, such as tick pyaemia (caused by Staphylococcus spp.) or Bibersteinia/Mannheimia septicaemia [64,65]. Other complications include abortion and reduced milk yield, impaired spermatogenesis in males, and reduced weight gain in young animals. Tick pyaemia, a wasting and crippling condition caused by Staphylococcus aureus, a common manifestation related to a primary A. phagocytophilum infection, is a severe challenge in some countries [64,66]. In contrast, secondary bacterial infections are not a significant part in the reported outbreaks of caprine TBF [18].
Several variants/strains of A. phagocytophilum have been characterized that seem to be involved in different natural enzootic cycles. Natural reservoirs of these variants are mostly unknown, although red deer seems to be involved as a reservoir host for strains causing TBF in sheep [64]. Current control strategies are based on the reduction of tick infestation by acaricides on tick pasture. This is mostly done be dipping or pour-on applications of pyrethroids [64].

Ehrlichia ruminantium (formerly Cowdria ruminantium)
E. ruminantium causes heartwater, a serious condition in ruminants with high mortality. The infection is transmitted by ticks in the genus Amblyomma, especially A. variegatum and A. hebraeum [67]. Heartwater is an endemic disease in domestic and some wild ruminants throughout sub-Saharan Africa, including islands near eastern Africa, but occurs also in the Caribbean [68]. There are four clinical forms of heartwater-peracute, acute, subacute and subclinical-whereas the acute form is most common [18]. The disease is characterized by the sudden onset of high fever, nervous signs, and rapid and abdominal breathing. E. ruminantium may cause high mortalities (more than 90% is recorded in sheep) related to breed, age and bacterial strains [69,70]. Subclinical and mild cases are common in young animals and in local indigenous breeds. Chronic infection has not been reported.
Although heartwater has been known for more than a century, it is still considered a major obstacle against the expansion and development of the livestock industry in southern Africa. The current methods for heartwater control include the use of acaricides to control the ticks, antibiotic prophylaxis, immunization by infection and treatment, farming with resistant breeds and establishment of endemic stability. Currently, there is no safe, user-friendly and reliable vaccine commercially available [71,72].