INTRODUCTION AND HISTORICAL BACKGROUND

The first report related to bovine besnoitiosis was published in 1884, when Cadéac described a skin disease in cattle he named elephantiasis (Cadéac, Reference Cadéac1884). In 1912, the French parasitologists Besnoit and Robin found that the disease described by Cadéac was caused by a parasite, and described it as sarcosporidiosis. However, they noted morphological differences from Sarcocystis blanchardi (the only known Sarcocystis species in cattle at that time, a synonym of Sarcocystis fusifirmis (Besnoit and Robin, Reference Besnoit and Robin1912). In the same year two papers commented on this discovery (Henry, 1912; Marotel, Reference Marotel1912). In 1916 Franco and Borges published an epidemiological and histological study of this cattle disease in Portugal covering a time span of 30 years. They proposed the genus Besnoitia and named the aetiological agent Besnoitia besnoiti (Franco and Borges, Reference Franco and Borges1916), leading to the introduction of the term besnoitiosis for the corresponding disease caused by these parasites.

Bovine besnoitiosis has been reported largely in sub-Saharan Africa (Bigalke and Prozesky, Reference Bigalke, Prozesky, Coetzer and Tustin2004) and Asia (Olias et al. Reference Olias, Schade and Mehlhorn2011). In Europe, after the earlier reports from France and Portugal, bovine besnoitiosis has been spreading on a larger scale (reviewed in Alvarez-Garcia et al. Reference Alvarez-Garcia, Frey, Ortega-Mora and Schares2013), such as in the south of Portugal (Cortes et al. Reference Cortes, Leitão, Vidal, Vila-Vicosa, Ferreira, Caeiro and Hjerpe2005, Reference Cortes, Nunes, Reis, Staubli, Vidal, Sager, Leitão and Gottstein2006a ), in Spain (Irigoien et al. Reference Irigoien, Del Cacho, Gallego, López-Bernad, Quílez and Sánchez-Acedo2000; Fernandez-Garcia et al. Reference Fernandez-Garcia, Risco-Castillo, Pedraza-Diaz, Aguado-Martinez, Alvarez-Garcia, Gomez-Bautista, Collantes-Fernandez and Ortega-Mora2009a , Reference Fernandez-Garcia, Alvarez-Garcia, Risco-Castillo, Aguado-Martinez, Marcen, Rojo-Montejo, Castillo and Ortega-Mora2010), France (Alzieu et al. Reference Alzieu, Cortes, Gottstein, Jacquiet, Dorchies, Schelcher and L'Hostis2007; Jacquiet et al. Reference Jacquiet, Liénard and Franc2010; Liénard et al. Reference Liénard, Salem, Grisez, Prévot, Bergeaud, Franc, Gottstein, Alzieu, Lagalisse and Jacquiet2011) and recent outbreaks have been reported in Germany (Mehlhorn et al. Reference Mehlhorn, Klimpel, Schein, Heydorn, Al-Quraishy and Selmair2009; Rostaher et al. Reference Rostaher, Mueller, Majzoub, Schares and Gollnick2010), Italy (Gollnick et al. Reference Gollnick, Gentile and Schares2010; Gentile et al. Reference Gentile, Militerno, Schares, Nanni, Testoni, Bassi and Gollnick2012) and Central Eastern Europe (Hornok et al. Reference Hornok, Fedák, Baska, Hofmann-Lehmann and Basso2014).

Frequently, the occurrence of bovine besnoitiosis coincides with the introduction of animals in farms. Basically these are males to be used for reproduction purposes, either avoiding consanguineous situations in herds or promoting heterosis. By buying sub-clinically infected animals that are introduced to farms without an appropriate diagnosis of infection for relevant parasitic diseases, owners are introducing a disease that can spread within the herd. When this occurs with Besnoitia-infected cattle, 10% of animals are expected to acquire the disease and to have lost their commercial value within the next 3 years (Pols, Reference Pols1960; Bigalke, Reference Bigalke1968). After 3 years the intra-herd prevalence is high (frequently higher than 80%) and from time to time an animal may develop clinical symptoms. When an infected herd has a high prevalence of sub-clinically infected animals, commonly the diseased animals are the naïve ones being introduced for reproduction purposes (Cortes et al. Reference Cortes, Leitão, Vidal, Vila-Vicosa, Ferreira, Caeiro and Hjerpe2005, Reference Cortes, Chagas e Silva, Baptista, Pereira, Leitão, Horta, Vasques, Barber, Marques and Ribeiro2006b ). Overall, this poses a significant economic burden in animal production.

Until some years ago, most veterinarians in Europe, when confronted with cattle suffering from excessive skin disorders, would not include bovine besnoitiosis in their differential diagnosis. This has changed. Information on the occurrence of bovine besnoitiosis in previously unaffected areas has been disseminated and schemes developed that aim at the prevention of the introduction of the parasite into infection-free herds. The European Food and Safety Authority (EFSA) has now recognized bovine besnoitiosis as a re-emerging disease and endemic in Europe (EFSA Journal, 2010).

BIOLOGICAL FEATURES OF THE GENUS BESNOITIA AND B. BESNOITI

The genus Besnoitia represents mandatory intracellular protozoan parasites belonging to the phylum Apicomplexa. The species in this genus are closely related to Neospora caninum, which causes abortion and stillbirth in cattle and neuromuscular disease in dogs, and the anthropozoonotic parasite Toxoplasma gondii. To the present date, no member of the genus Besnoitia has been found to be infective for humans. At least ten Besnoitia species have been recognized so far worldwide, most of them have been reported from marsupials and micro mammals, where the cysts are more prevalent internally, mainly in the mesenterium, and for these species the cat has been identified as the definitive host. In contrast, tissue cysts with a high tropism for the skin are presented during infections with Besnoitia benneti in equines (van Heerden et al. Reference Van Heerden, Els, Raubenheimer and Williams1993; Elsheikha et al. Reference Elsheikha, Mackenzie, Rosenthal, Marteniuk, Steficek, Windsor, Saeed and Mansfield2005), Besnoitia tarandi in deer (Ayroud et al. Reference Ayroud, Leighton and Tessaro1995; Dubey et al. Reference Dubey, Sreekumar, Rosenthal, Vianna, Nylund, Nikander and Oksanen2004; Ducrocq et al. Reference Ducrocq, Beauchamp, Kutz, Simard, Elkin, Croft, Taillon, Cote, Brodeur, Campbell, Cooley, Cuyler and Lair2012) and B. besnoiti in cattle.

Those Besnoitia species found in rodents and lagomorphs from the American continent (Besnoitia neotomofelis, Besnoitia darlingi and Besnoitia oryctofellisi) have been well characterized, their life cycles have been described and the cat was found to act as the definitive host (Olias et al. Reference Olias, Schade and Mehlhorn2011). Besnoitia tarandi and Besnoitia bennetti were isolated in Europe and USA respectively, and B. besnoiti was isolated from infected cattle in South Africa, Israel and different European countries, and for these three species, the respective definitive host has not been identified to date. Thus, it is not known where and how, or if, in their life cycle sexual reproduction takes place. There have been several attempts to generate B. besnoiti oocysts in cats. There is only one report which claims that oocyst formation has occurred in cats fed on cattle tissues supposedly infected with B. besnoiti (Peteshev et al. Reference Peteshev, Galuzo and Polomoshov1974). However, others have not confirmed these results (Dubey, Reference Dubey1976; Soulsby, Reference Soulsby1982; Rommel, Reference Rommel1989), and attempts to infect domestic cats by feeding tissue cysts have failed (Diesing et al. Reference Diesing, Heydorn, Matuschka, Bauer, Pipano, de Waal and Potgieter1988; Ng'ang'a and Kasigazi, Reference Ng'ang'a and Kasigazi1994; Ayroud et al. Reference Ayroud, Leighton and Tessaro1995; Basso et al. Reference Basso, Schares, Gollnick, Rütten and Deplazes2011).

All phylogenetic analyses on the genus Besnoitia have been based on the availability of ITS1 gene sequences. Although sequence information is not entirely complete for all species, it was possible to identify a Besnoitia genus-specific cluster within the ITS1 region which, by multiple alignments, was demonstrated to be independent from other apicomplexans. Phylogeny suggests that the Besnoitia genus is comprised of two distinct groups. One group includes all Besnoitia isolates from cattle, equines and deer, while the other contains Besnoitia isolates from rodents and lagomorphs from the American continent (Kiehl et al. Reference Kiehl, Heydorn, Schein, Al-Rasheid, Selmair, Abdel-Ghaffar and Mehlhorn2010). The latter exhibits a higher degree of ITS1 sequence variation. This may be related to the fact that the diploid phase contributes to a larger extent during differentiation and the life cycle, with a higher chance of recombination occurring. The ITS regions have been used both to distinguish species (Coleman and Mai, Reference Coleman and Mai1997) and to differentiate between populations of the same species (Rinder et al. Reference Rinder, Rausch, Takahashi, Kopp, Thomschke and Loscher1997). Isolates from infected cattle in Portugal, Spain, Germany and France share the same clinical manifestations and histopathological characteristics of the reported B. besnoiti isolated in South Africa and Israel (Basson et al. Reference Basson, McCully and Bigalke1970; Diesing et al. Reference Diesing, Heydorn, Matuschka, Bauer, Pipano, de Waal and Potgieter1988), and the ITS1 transcribed region shows a high level of conservation in these species (Kiehl et al. Reference Kiehl, Heydorn, Schein, Al-Rasheid, Selmair, Abdel-Ghaffar and Mehlhorn2010). The ITS1 transcribed region-based grouping of Besnoitia species does not seem to be related to the geographic origin of the respective isolates but is more likely to be connected with the nature of the respective intermediate hosts.

For B. besnoiti in cattle, the oral route of infection through oocysts shed by a definitive host, as established for T. gondii and cats, or N. caninum and dogs, has not been identified to date. However, it was demonstrated that one form of transmission occurs by blood feeding insects such as horse flies and the stable fly, Stomoxys calcitrans (Bigalke, Reference Bigalke1968), therefore simply bypassing the sexual cycle and promoting clonality of B. besnoiti. This was also suggested by comparison of the sequences of ITS1-transcribed regions of B. besnoiti from different geographic regions, showing a lower degree of sequence variation for mechanically transmitted Besnoitia species (Kiehl et al. Reference Kiehl, Heydorn, Schein, Al-Rasheid, Selmair, Abdel-Ghaffar and Mehlhorn2010). Nevertheless, it is important to investigate more Besnoitia isolates from different species and geographic areas, and respective molecular characterization will allow more insights related to transmission and host specificity. It is, however, conceivable that besnoitiosis in cattle simply represents a disease caused by accidental infection, and that the actual life cycle of B. besnoiti has not yet been discovered because it takes place in wildlife (Franco and Borges, Reference Franco and Borges1916; Kiehl et al. Reference Kiehl, Heydorn, Schein, Al-Rasheid, Selmair, Abdel-Ghaffar and Mehlhorn2010; Basso et al. Reference Basso, Schares, Gollnick, Rütten and Deplazes2011).

MORPHOLOGICAL FEATURES OF B. BESNOITI

Besnoitia besnoiti tachyzoites represent the proliferative stage responsible for acute disease. They are 6–7·5×2·5–3·9 μm (Reis et al. Reference Reis, Cortes, Viseu, Fazendeiro, Leitão and Soares2006) and can be cultured in vitro employing various mammalian cell types, using similar techniques as for N. caninum and T. gondii tachyzoites (Shkap et al. Reference Shkap, Pipano and Greenblatt1987b ). By light microscopy, B. besnoiti tachyzoites cannot be differentiated from Neospora or Toxoplasma tachyzoites. By TEM, the hallmarks of apicomplexans are clearly visible. These include the invasion-relevant secretory organelles such as micronemes, rhoptries and dense granules, and the conoid and the microtubular cytoskeleton underlying the plasma membrane (see Fig. 1). As for T. gondii and N. caninum, these tachyzoites can reside within a wide range of host cells, and they occupy a cytoplasmic compartment called the parasitophorous vacuole, which is surrounded by a parasitophorous vacuole membrane. Proliferation occurs through endodyogeny, with a replication rate of tachyzoites, depending on cell lines, from 0·14 and 20 tachyzoites per hour (Schares et al. Reference Schares, Basso, Majzoub, Cortes, Rostaher, Selmair, Hermanns, Conraths and Gollnick2009). As with other apicomplexans, B. besnoiti tachyzoites enter their host cells with the apical part first, most likely employing similar mechanisms as described for T. gondii involving actin/myosin motor proteins. However, clear differences exist with respect to the structure and dynamics of tubulin polymers in Toxoplasma and Besnoitia, and with respect to their interaction with the host microtubular cytoskeleton once host cell invasion is achieved (Reis et al. Reference Reis, Cortes, Viseu, Fazendeiro, Leitão and Soares2006). In addition, the two species also differentially modulate the host cell centrosome and the Golgi apparatus (see paper by Cardoso et al. in this Special Issue).

Fig. 1.

Besnoitia besnoiti tachyzoites. (A) and (B) show scanning electron micrographs of tachyzoites (T) establishing contact with the surface of Vero cells. Arrows point to parasites in the process of host cell invasion. Following entry, tachyzoites proliferate rapidly within a parasitophorous vacuole (C–E). Arrows in (E) point towards the parasitophorous vacuole membrane. E is a higher magnification view of (D). Micronemes (mic), dense granules (dg), rhoptries (dg) and the parasite mitochondrion are clearly visible, n indicates the nucleus, and the apicoplast (Ap) is visible in one of the tachyzoites in (E). Note the parasitophorous vacuole membrane indicated by arrows. Bars in A = 1·8 μm; in B = 3·6 μm; in C = 1 μm; in D = 1·9 μm; in E = 0·65 μm.

As with the other apicomplexans, tissue cysts of B. besnoiti harbouring bradyzoites persist within chronically infected animals for extended periods of time. Bradyzoites are 6·0–7·5×1·9–2·3 μm (Dubey et al. Reference Dubey, Shkap, Pipano, Fish and Fritz2003), and they form spectacular tissue cysts often containing hundreds of parasites. These cysts are characterized by two distinct cyst walls (Fig. 2). The inner cyst wall is, similar to T. gondii and N. caninum, located intracellularly within a large multinuclear host cell, surrounded by the membrane of the parasitophorous vacuole, and is most likely synthesized by the parasite. The second cyst wall surrounds the entire host cell and is 10–12 μm in thickness (Dubey et al. Reference Dubey, Shkap, Pipano, Fish and Fritz2003; Cortes et al. Reference Cortes, Reis, Waap, Vidal, Soares, Marques, da Pereira, Fazendeiro, Ferreira, Caeiro, Shkap, Hemphill and Leitão2006c ). The outer cyst wall is comprised of collagenous material (Ayroud et al. Reference Ayroud, Leighton and Tessaro1995) and seems to be a product of a physiological reaction on the part of the host (Dubey et al. Reference Dubey, van Wilpe, Blignaut, Schares and Williams2013). In any case, it is this double-layered cyst wall which clearly distinguishes B. besnoiti from its related apicomplexans.

Fig. 2.

Besnoitia besnoiti bradyzoites. (A) Shows a histological section cut through a biopsy of the dermis and epidermis of a cow with chronic besnoitiosis. Tissue cysts with a double-layered cyst wall are present at a high density. (B) Shows a section through the interior part of a tissue cyst, with numerous bradyzoites embedded in a granular matrix (m). Note the high numbers of micronemes (m) and the parasite nucleus (n), which is located at the posterior end of the parasite. (C) and (D) are sections showing peripheral areas of tissue cysts. Bradyzoites at the cyst periphery are marked with a b, and the asterisk indicates a dead and partially lysed bradyzoite. The inner cyst wall (icw) is located within a host cell and presents a rather amorphous appearance. Arrows in (C) and (D) point towards the parasitophorous vacuole membrane. The outer cyst wall (ocw) surrounds the entire host cell and is a structure rich in densely packed filaments. In (D) a flattened host cell nucleus (hcn) is visible in the space between inner and outer cyst wall (hcc = host cell cytoplasm). Bars in A = 600 μm; in B = 0·9 μm; in C and D = 1·5 μm.

AETIOLOGY, PATHOGENESIS AND CLINICAL DIAGNOSIS OF BOVINE BESNOITIOSIS

Bovine besnoitiosis may have two different presentations: an acute stage occurring 11–13 days after infection and lasting 6–10 days, followed by a chronic stage, which develops while the animals recover from the acute stage, and is lifelong (Pols, Reference Pols1960; Bigalke, Reference Bigalke1968).

The acute stage is characterized by rapid proliferation of tachyzoites and subsequent immunopathology and tissue destruction, all within 1–2 weeks post-infection. Tachyzoites proliferate in macrophages, fibroblasts and endothelial cells within blood vessels. The result is vasculitis and thrombosis, especially of capillaries and small veins in the dermis, subcutis, fascia, testes and upper respiratory mucosae (Basson et al. Reference Basson, McCully and Bigalke1970). Acute stage clinical signs are increased body temperature (above 40 °C), intensive respiratory disorder, increased heart rates, serous nasal and ocular discharges, anorexia, generalized weakness, loss of milk production, reluctance to move, rapid loss of weight and declining body condition due to diminished food uptake, swelling of superficial lymph nodes, generalized oedema of the skin, acute orchitis with swollen, painful testes and, in some cases, anasarca (Schulz, Reference Schulz1960; McCully et al. Reference McCully, Basson, van Niekerk and Bigalke1966; Cortes et al. Reference Cortes, Leitão, Vidal, Vila-Vicosa, Ferreira, Caeiro and Hjerpe2005). Inspiratory dyspnoea may result from inflammation of the upper respiratory mucosae (McCully et al. Reference McCully, Basson, van Niekerk and Bigalke1966). The acute stage does not always lead to death, but these processes can result in dramatically decreased body condition scores (Cortes et al. Reference Cortes, Leitão, Vidal, Vila-Vicosa, Ferreira, Caeiro and Hjerpe2005). In pregnant animals, this pathophysiological disorder can lead to abortion (Pols, Reference Pols1960; Juste et al. Reference Juste, Cuervo, Marco and Oregui1990).

One to 2 weeks after the onset of acute stage symptoms, the oedema reduces and the infection reaches the chronic stage, which is characterized by the formation of tissue cysts of up to 0·5 mm in diameter by slowly proliferating bradyzoites, which can persist for several years in different tissues of the host (Fig. 2A). These characteristic cysts form especially in cutaneous and subcutaneous tissues, and in intermuscular fascia (McCully et al. Reference McCully, Basson, van Niekerk and Bigalke1966; Basson et al. Reference Basson, McCully and Bigalke1970). A low-grade, intermittent febrile reaction may be observed, causing anorexia and weight loss. Dermal lesions, presenting different extents of hair loss, are always present during chronic disease. These consist of rather dramatic thickening, hardening and folding or wrinkling of the skin, especially around the neck, shoulders and rump, always accompanied by hyperkeratosis, hyperpigmentation and alopecia (Pols, Reference Pols1960). The thickening of the skin is caused by scleroderma (Basson et al. Reference Basson, McCully and Bigalke1970). Scleroderma and alopecia are permanent disfigurements in surviving animals (Bigalke, Reference Bigalke1960). There may also be pronounced thickening of the limbs, and locomotion may be difficult and painful (Pols, Reference Pols1960). A mucopurulent nasal discharge may be accompanied by inspiratory dyspnoea (McCully et al. Reference McCully, Basson, van Niekerk and Bigalke1966). Small cysts that are visible by eye are usually apparent upon close visual inspection of the scleral conjunctiva and vulvae (Rostaher et al. Reference Rostaher, Mueller, Majzoub, Schares and Gollnick2010; Frey et al. Reference Frey, Gutiérrez-Expósito, Ortega-Mora, Benavides, Marcén, Castillo, Casasús, Sanz, García-Lunar, Esteban-Gil and Álvarez-García2013; see Fig. 3), a feature that is of considerable value in clinical diagnosis (Sannusi, Reference Sannusi1991).

Fig. 3.

Selected clinical features of bovine besnoitiosis. (A) Shows an udder of a chronically infected cow presenting wounds in the teats, resulting from repeated lesions caused by nursing the calf. Individual and/or clusters of cysts can be detected by eye in the conjunctive (B) and in the vulva (C).

All cattle breeds, both sexes and animals of all ages can be affected, except that acute and chronic disease seldom occurs in calves of less than 6 months of age (Bigalke, Reference Bigalke1968; Alzieu et al. Reference Alzieu, Cortes, Gottstein, Jacquiet, Dorchies, Schelcher and L'Hostis2007). Animals with poor body condition are usually culled without any commercial value or die in the field. In cases where the chronic stage does not dramatically decrease the health status of the animal, the parasite will persist and will present a large number of cysts in any part of the skin during the entire lifespan, even if the animal recovers body condition. Cows at this stage are still fertile and frequently become pregnant and give birth. There are no reports of vertical transmission of B. besnoiti. The disease, however, has a negative impact on milk production, and this often negatively affects calves in their growth capacity (Cortes et al. Reference Cortes, Reis, Waap, Vidal, Soares, Marques, da Pereira, Fazendeiro, Ferreira, Caeiro, Shkap, Hemphill and Leitão2006c ). However, due to the high number of skin cysts in the mucosae of teats and udder, and the loss of elasticity, the teats will present large wounds and will bleed (Fig. 3). Newborn calves do not have antibodies against B. besnoiti, and receive them through suckling from the dam, soon acquiring titres comparable to the dam up to the age of 6–8 months (Shkap et al. Reference Shkap, Pipano, Marcus and Krigel1994).

Infected bulls present the same symptoms in the acute and chronic stages of the disease as cows. Severely affected bulls often develop irreversible intratesticular lesions of vasculitis, focal necrosis, sclerosis and atrophy, which usually result in permanent infertility (Kumi-Diaka et al. Reference Kumi-Diaka, Wilson, Sannusi, Njoku and Osoru1981). The case fatality rate during the chronic stage is usually in the order of 10% (Pols, Reference Pols1960).

Although some of the typical clinical aspects of bovine besnoitiosis have been well described, the diagnosis based on clinical signs is difficult, and the disease may be misdiagnosed as a fungal infection, rinderpest, scabies, mineral deficiency, photosensitivity or even blue tongue virus infection, and differential laboratory diagnostics need to be further improved.

LABORATORY DIAGNOSIS

Presently, different diagnostic methods are available for the detection of B. besnoiti infection in cattle. These include direct detection of the parasite and/or its DNA in tissue samples (histopathology, PCR) and indirect detection based on serology. Application of these methods or combinations thereof depends on the clinical status of the animal and the status of the herd it comes from.

TRANSMISSION OF B. BESNOITI TO CATTLE

Cattle are the only known intermediate host of B. besnoiti. Elucidation of the entire life cycle would be important to define the role of the putative definitive host in transmission, and would aid in the development of appropriate prophylaxis and control measures. It is not even known, however, whether a definitive host exists, and if so, how important it is in transmitting the parasite. In fact, it has been suggested that some T. gondii populations represent clonal lineages, indicating that transmission is not dependent on the diploid infective stage in the definitive host (Johnson, Reference Johnson1997).

Similar to other cyst-forming coccidians, natural exposure of cattle to sporulated oocysts suggests the presence of a definitive host in the immediate environment (most likely a carnivore) or an infective feed source (infected or contaminated with tachyzoites or bradyzoites of B. besnoiti) to which the definitive host has access (Tenter and Johnson, Reference Tenter and Johnson1997). On many farms where bovine besnoitiosis is endemic, dead cattle in farms are conscientiously disposed of by rendering, incineration, deep burial or other means that do not allow for recycling of infectious materials into potential definitive hosts. On the other hand, it is possible that a sylvatic cycle exists and as a consequence oocysts could be produced by a definitive host in wildlife. That this possibility is viable was suggested in South Africa (Basson et al. Reference Basson, van Niekerk, McCully and Bigalke1965, Reference Basson, McCully and Bigalke1970; McCully et al. Reference McCully, Basson, van Niekerk and Bigalke1966; Bigalke et al. Reference Bigalke, van Niekerk, Basson and McCully1967, Reference Bigalke, Schoeman and McCully1974), where B. besnoiti has been shown to affect wildebeest, kudu and impala. However, Bigalke et al. (1967) registered differences among isolates from wildebeest, impala and cattle, and concluded that they should be regarded as distinct strains or biologically different isolates of B. besnoiti. There is also serological evidence for B. tarandi and/or B. besnoiti infection in wild ruminants in Canada (caribou, reindeer, mule deer and musk ox with similar characteristic clinical signs and lesions) and in Spain (red deer and roe deer) (Gutiérrez-Expósito et al. Reference Gutiérrez-Expósito, Ortega-Mora, Gajadhar, García-Lunar, Dubey and Alvarez-García2012, Reference Gutiérrez-Expósito, Ortega-Mora, Marco, Boadella, Gortázar, San Miguel-Ayanz, García-Lunar, Lavín and Alvarez-García2013), but the meaning of these observations for the explanation of the transmission of B. besnoiti to cattle remains to be elucidated.

Within endemic areas there is evidence that biting flies can mechanically transmit B. besnoiti (Pols, Reference Pols1960; Alzieu et al. Reference Alzieu, Cortes, Gottstein, Jacquiet, Dorchies, Schelcher and L'Hostis2007; Liénard et al. Reference Liénard, Salem, Grisez, Prévot, Bergeaud, Franc, Gottstein, Alzieu, Lagalisse and Jacquiet2011, Reference Liénard, Salem, Jacquiet, Grisez, Prévot, Blanchard, Bouhsira and Franc2013). Outbreaks of bovine besnoitiosis mostly occur during seasons of the year when biting flies are active. Several biting insects such as Stomoxys, Glossina and Chrysops have been implicated in the mechanical transmission of infection (Pols, Reference Pols1960; Bigalke, Reference Bigalke1968). They transport bradyzoites in their mouthparts and abdominal contents from one host to the other and deposit them onto and/or into the skin. Since tachyzoites have been demonstrated in lacrimal secretions (Bigalke, Reference Bigalke1968; Cortes et al. Reference Cortes, Ferreira, Silva, Vidal, Serra and Caeiro2003), there is a possibility that non-biting flies, such as Musca autumnalis and Musca domestica, and other insects, might also be capable of mechanical transmission by having access to B. besnoiti in the lacrimal fluid. Nevertheless mechanical transmission would in this case result in parasite transfer from disrupted cysts in wounds of infected animals to wounds of susceptible ones, through direct contact of legs and licking mouthparts, but this is expected to be extremely rare in field conditions (Liénard et al. Reference Liénard, Salem, Jacquiet, Grisez, Prévot, Blanchard, Bouhsira and Franc2013). Introduction of sub-clinically or clinically infected animals into herds that had not been exposed to Besnoitia before may often result in an outbreak of disease. Thus, acquisition of infected animals should be avoided. Conversely, the fact that clinical signs occurred rapidly in animals that have been introduced into herds known to be infected suggested that mechanical transmission occurred within a short period, most likely due to a high population of potential mechanical vectors (Pols, Reference Pols1960; Bigalke, Reference Bigalke1968; Liénard et al. Reference Liénard, Salem, Grisez, Prévot, Bergeaud, Franc, Gottstein, Alzieu, Lagalisse and Jacquiet2011). In addition, B. besnoiti can also be transmitted iatrogenically during prophylactic or treatment procedures within the herd (Pols, Reference Pols1954, Reference Pols1960; Bigalke, Reference Bigalke1968). Finally, another mode, although speculative, might be a direct transmission from animal to animal, e.g. via mucosal contact by licking. If a chronically infected host presents many mucosal surface cysts such as frequently seen on the eye (and then maybe also other mucosal surfaces accessible for licking), contact with mechanically released bradyzoites from one animal to the oral mucosa of another animal may be a possibility.

TREATMENT OPTIONS FOR BOVINE BESNOITIOSIS

The first reported attempts for the treatment of bovine besnoitiosis were based on a single intravenous injection of 30 mL of a 1% solution of formalin during the acute stage of the disease (Herin, Reference Herin1952). For the treatment of chronically infected animals, the same author recommended administering 30–40 mL Lugol's iodine solutions 5 times intravenously at intervals of 4–7 days. However, these treatment approaches were not further developed, due to obvious adverse events associated with such treatments. Other treatment approaches were investigated in rabbits, including the administration of pentamidine, aureomicin, formalin, sodium iodide, sulfamerazine, mycostatin and terramycin, but were not successful (Pols, Reference Pols1960). Shkap et al. (Reference Shkap, De Waal and Potgieter1985b ) tested several compounds in rabbits experimentally infected with B. besnoiti tachyzoites obtained from cell culture. Oxytetracycline, given at 30 mg kg⁻¹ i.m. simultaneously with infection, prevented the development of orchitis. All infected animals showed a transient febrile reaction, and the authors concluded that oxytetracycline had some therapeutic potential against B. besnoiti and that rabbits are a suitable model for therapeutic trials of acute disease. Subsequent studies were performed in vitro and in experimentally infected gerbils. Drugs investigated in gerbils included oxytetracycline, sulfonamides, trimethoprim, halofunginone, diaminazene aceturate and pentamidine. Of these compounds, only oxytetracycline prevented death in gerbils, but only if administered at the time of infection (Shkap et al. Reference Shkap, Pipano and Ungar-Waron1987a ).

More recently, the nitro-thiazolide nitazoxanide and a range of bromo-derivatives were shown to inhibit B. besnoiti tachyzoite proliferation in Vero cells, and induced severe ultrastructural alterations. Bromo-derivatives were found to be equally effective as nitro-compounds, indicating that they could be a safer alternative to compounds containing a potentially harmful nitro group (Cortes et al. Reference Cortes, Mueller, Esposito, Leitão, Naguleswaran and Hemphill2007b ). Another in vitro study demonstrated that a series of new-generation pentamidine derivatives, arylimidamides, exhibit profound activity against B. besnoiti tachyzoites, but corresponding studies in animals have not yet been undertaken (Cortes et al. Reference Cortes, Muller, Boykin, Stephens and Hemphill2011).

VACCINES

Live vaccines have been used in South Africa and in Israel. In South Africa, the vaccine is based on tachyzoites of an isolate from blue wildebeest grown in cell cultures (Bigalke et al. Reference Bigalke, Schoeman and McCully1974) and is recommended for use in weaners and older animals. It protects cattle against clinical besnoitiosis, although it does not entirely prevent sub-clinical infection (Bigalke and Prozesky, Reference Bigalke, Prozesky, Coetzer and Tustin2004). The vaccine in use in Israel contains live attenuated parasites derived from cell cultures (Pipano, Reference Pipano1997) and there are no data in the scientific literature that allows for any judgement on its efficacy and safety. However, the use of any of these vaccines is geographically limited. Live-attenuated vaccines pose risks of introducing the parasite into uninfected herds and of inducing carriers among the vaccinated animals. This is of particular concern when, as in the case of B. besnoiti, the knowledge on the biology, transmission and life cycle of the parasite is scarce. This lack of knowledge has hampered the development of new vaccines. Little is known about the chain of infection and the infective stage and portal of entry for bovines under natural conditions. These aspects are of major relevance for the development and evaluation of immunization protocols and, in a broader sense, for the development of studies towards a better understanding of the bovine immune response against B. besnoiti infection.

Also crucial for the development of new vaccines against the disease is the characterization of the antigenic mosaic of the parasite. An important contribution can be taken from the studies mentioned above on the development of diagnostic tests based on western blot. The pattern of antigens with high diagnostic value (e.g. Schares et al. Reference Schares, Langenmayer, Scharr, Minke, Maksimov, Maksimov, Schares, Bärwald, Basso, Dubey, Conraths and Gollnick2013) certainly includes those antigens that are serologically immunodominant, and potentially suitable as vaccine targets. However, their molecular identification and characterization remains largely incomplete. Njagi et al. (Reference Njagi, Entzeroth, Nyaga and Musoke2004) characterized a panel of four monoclonal antibodies, obtained from mice immunized with live tachyzoites, two of which have been shown to significantly inhibit the invasion of cell culture monolayers by B. besnoiti tachyzoites. However, no subsequent studies were published and the antigens recognized by these monoclonal antibodies remain to be identified.

So far, the only B. besnoiti protein characterized and expressed in a heterologous system has been disulphide isomerase (BbPDI) (Marcelino et al. Reference Marcelino, Martins, Morais, Nolasco, Cortes, Hemphill, Leitão and Novo2011). Toxoplasma gondii and N. caninum had been previously shown to express highly homologous molecules (Meek et al. Reference Meek, Back, Klaren, Speijer and Peek2002a , Reference Meek, Back, Klaren, Speijer and Peek b ; Naguleswaran et al. Reference Naguleswaran, Alaeddine, Guionaud, Vonlaufen, Sonda, Jenoe, Mevissen and Hemphill2005) that were shown to be targeted, respectively, by human (Meek et al. Reference Meek, Back, Klaren, Speijer and Peek2002a , Reference Meek, Back, Klaren, Speijer and Peek b ) and bovine (Liao et al. Reference Liao, Ma, Bannai, Lee, Xie, Tang, Zhang, Xuan and Fujisaki2006) IgA. The N. caninum PDI was demonstrated to be important for the host cell invasion (Naguleswaran et al. Reference Naguleswaran, Alaeddine, Guionaud, Vonlaufen, Sonda, Jenoe, Mevissen and Hemphill2005) and turned out to be an interesting candidate for vaccine development (Debache et al. Reference Debache, Guionaud, Alaeddine and Hemphill2010). Interestingly, conversely to what has been observed for N. caninum (Shin et al. Reference Shin, Lee, Shin, Kim, Lee, Kim, Jang, Gershwin, Kim, Kim, Kim, Suh and Jung2004, Reference Shin, Shin, Kim, Lee, Yang, Palaksha, Youn, Kim, Kim, Marsh, Lakritz and Jung2005), a recent study tackling the characterization of the immunome of B. besnoiti tachyzoites using pooled sera from seven naturally infected cattle (Garcia-Lunar et al. Reference Garcia-Lunar, Regidor-Cerrillo, Gutiérrez-Expósito, Ortega-Mora and Alvarez-García2013b ) has not confirmed BbPDI as a serologically dominant antigen. In this study, out of the 20 immunogenic proteins identified based on homology with sequences available in databases from other members of the Toxoplasmatinae sub-family, were four proteins related to energy metabolism (fructose-1,6-bisphosphate aldolase, LDH, pyruvate kinase and ENO), three heat shock proteins (Hsp60, Hsp70 and Hsp90), and actin and profilin.

Fernandez-Garcia et al. (Reference Fernandez-García, Alvarez-García, Marugán-Hernández, García-Lunar, Aguado-Martínez, Risco-Castillo and Ortega-Mora2013) reported on the identification of antigens that are differentially expressed in B. besnoiti tachyzoites and bradyzoites. They performed comparative 2D-gel electrophoresis on extracts of both stages, followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively, and 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, five up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and five up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. These proteins can now be further studied with regard to their role in host-cell parasite interactions, tachyzoite-bradyzoite transition, and in terms of a putative role as drug targets or vaccine candidates.

Clearly, the contribution of these studies for the development of safer subunit vaccines is hampered by two main obstacles. First, the scarce knowledge currently available on the molecular aspects of this parasite, and second the absence of a good experimental animal model. Thus, B. besnoiti genome and transcriptome sequencing as a basis for the systematic molecular analysis of its biology must be a priority, as well as the development of an experimental bovine model that allows assessment of the efficacy of potential vaccine candidates directly in the most relevant target host.