Volume 21 • Number 6 • 2017 Heartwater in and Small Ruminants

Dr. P.H. Mapham, BVSc (Hon) Dr. J.H. Vorster, BVSc, MMedVet(Path) 78 Craigie Drive PO Box 13624 Pietermaritzburg, 3201 Cascades, 3202 Tel no: 033 347 2259 Tel no: 033 342 5104 Cell No: 082 771 3227 Cell no: 082 820 5030 E-mail: [email protected] E-mail: [email protected]

Introduction mals. According to the OIE it is not known for how According to Dr PG Marais heartwater was first diag- long wild or domestic ruminants can be a source nosed in the North-eastern Transvaal in 1838 (now of infection for in nature, but it may be many Mpumalanga) and he makes the interesting com- months. Ticks are a robust reservoir of E. ruminan- ment that the actual origin of the disease is unknown tium, and infection can persist in them for at least and that it is doubtful whether the disease is indig- 15 months. enous to South Africa. Nevertheless, over the years heartwater has caused considerable loss to live- Of the 13 species capable of transmitting the dis- stock producers and frustration to veterinarians trying ease, A. variegatum (tropical bont ) is by far the to control it. Recently reports of it occurring where it most important because it is the most widespread. wasn’t found previously have been emerging and Other major vector species are the bont tick A. it is appropriate for veterinarians to review this sig- hebraeum (in southern Africa), A. gemma and A. nificant disease. Much of the following article was lepidum (in Somalia, East Africa and the Sudan). A. adapted from OIE information astrion (mainly feed on buffalo) and A. pomposum (distributed in Angola, Congo [Dem. Rep. of the] Aetiology and Central African Republic) are also natural vec- Heartwater is caused by ruminantium (for- tors of the disease. merly Cowdria ruminantium) which is of the order , Family Anaplasmataceae. These are Four other African ticks, A. sparsum (feed on reptiles small, gram negative, pleomorphic cocci, which and buffalo mainly), A. cohaerans (feed on African are obligate intracellular parasites. There are diverse buffalo), A. marmoreum (adults occur on tortoises strains of E. ruminantium which vary in virulence and and immature stages on ) and A. tholloni have a high level of genomic plasticity. Several dif- (adults feed on elephants) experimentally transmit ferent genotypes may coexist in a geographical heartwater. Three North American species of Ambly- areas, and may recombine to form new strains ac- omma ticks also experimentally transmit heartwater: cording to the OIE. The heartwater organism is ex- A. maculatum (the Gulf Coast tick), A. cajennense tremely fragile and cannot persist outside of a host (the Cayenne tick) and A. dissimile, but none of these for more than a few hours. ticks has been incriminated so far in natural transmis- sion of heartwater. A. maculatum is widely distrib- Epidemiology uted in the eastern, southern, and western U.S., and Heartwater occurs only where its tick feeds on ungulates (cattle, , goats, horses, vectors are present and infection depends on the pigs, bison, donkeys, mules, white-tailed deer, sam- epidemiological triad of the tick vector, causative bar deer and axis deer), various carnivores, rodents agent, and hosts. The tick vector is sub- and lagomorphs, marsupials, birds, and reptiles. A. ject to seasonal, environmental and management maculatum was shown to be as efficient as A. he- variables. The principal method of bringing the braeum, and was susceptible to a wide range of E. disease into an area is by introduction of infected ruminantium strains. A. cajennense has host prefer- ticks or carrier animals. Ticks become infected by ence similar to A. maculatum but is not as widely feeding on acutely ill or sub clinically infected ani- distributed and is a less efficient heartwater vector.

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A. dissimile feeds on reptiles and amphibians. laphus tragocamelus), fallow deer (Damadama), Amblyomma ticks are three-host ticks whose life Himalayan tahr (Hemitragus jemlahicus), barbary cycles may take from 5 months to 4 years to com- sheep (Ammotragus lervia), mouflon (Ovis aries), plete. Because the ticks may pick up the infection blackbuck (Antilope cervicapra), and white and as larvae or nymphs and transmit it as nymphs or as black rhinoceros. adults, the infection can persist in the tick for at least 15 months. Infection does not pass transovarially. Wildlife used in farming, seem to be the main wild ru- While transmission of heartwater can be by adult and minant species in which heartwater can have a sig- nymphal ticks in the field, in general adults prefer to nificant economic impact. The OIE states that it was feed on cattle and nymphs on sheep and goats. once believed that the guinea fowl and leopard tor- Cattle egrets have been implicated in the dispersal toises were the nonruminant hosts of E. ruminantium, of Amblyomma ticks in the Caribbean. but recent data have confirmed that these species are not susceptible and do not transmit to vector The causative agent, E. ruminantium multiplies in ticks that usually feed on them. The scrub hare’s sus- vascular endothelial cells throughout the body to ceptibility to infection is also not fully substantiated. cause vascular compromise with varying virulence. Although the striped mouse and the multimammate Because of its extreme fragility, it is only able to be mouse have been shown to be susceptible to E. transmitted by ticks in the presence of infected ani- ruminantium, they are not hosts of the vector ticks mals. Vertebrate hosts include all domestic and wild and are not believed to play a role in the epidemiol- ruminants, but indigenous domestic and wild rumi- ogy of heartwater. Some laboratory inbred strains of nants appear to be less susceptible than other do- mice have been shown to be susceptible to E. rumi- mestic ruminants. Heartwater causes severe disease nantium and have assisted in defining disease and in cattle, sheep, and goats, with milder disease in immune mechanisms, but these are not indicated some indigenous African breeds of sheep and as important in disease maintenance. goats, and inapparent disease in several species of antelope indigenous to Africa. Bos indicus (Zebu) Transmission cattle breeds are in general more resistant than Heartwater is transmitted trans-stadially by ticks of the Bos taurus (European) breeds. Up to 80% of merino genus Amblyomma, which are biological vectors of sheep may die, but the mortality rate can be only heartwater. Ticks become infected by feeding on an 6% in Persian or Afrikaner sheep. infected vertebrate host during the febrile reaction. Whole blood or plasma from the vertebrate is infec- Angora and Saanen goats are also very suscepti- tive, but the highest levels of the agent occur during ble to heartwater, while indigenous goats in certain the second or third day of . areas may show a significant level of resistance. Heartwater has caused mortality in the African buf- Heartwater can apparently be transmitted vertically falo (Syncerus caffer) in some situations while deaths and through colostrum of carrier dams containing in springbok (Antidorcas marsupialis) in South Africa infected cells (reticuloendothelial cells and macro- have been attributed to heartwater. Other species phages). that have been shown to be susceptible are the blesbok (Damaliscus albifrons), the Transmission can also occur by intravenous inocula- (Connochaetes gnu), the eland (Taurotragus oryx tion of blood, tick homogenates or cell culture ma- oryx), giraffe (Giraffe camelopardalis), greater kudu terial containing E. ruminatium. (Tragelaphus strepsiceros), sable antelope (Hippotra- gus niger), sitatunga (Tragelaphus spekii), steenbok Horak et al reported the recovery of adult A. hebrae- (Raphicerus campestris), and lechwe (Kobus leche um from rhinoceroses at four localities in the Free kafuensis). It is believed that these species may serve State constituting a new distribution record. They be- as reservoirs of heartwater and the disease in these lieve that the ticks were introduced into the province animals is usually mild or undetectable. According on the animals concerned and warn that should A. to the OIE there is one report of an African elephant hebraeum become established in the Free State dying of heartwater, but this animal was also infect- where the climate and vegetation may be suitable ed with anthrax. for its survival, it will be nearly impossible to eradicate because of the approximately 15 000 eggs laid by Many non-African ruminants are experimentally engorged females. Should cattle or antelope spe- susceptible to heartwater including the Timor deer cies that are asymptomatic carriers of the organism (Cervus timorensis) and chital (Axis axis) of southern be translocated from regions in which heartwater Asia, and white-tailed deer (Odocoileus virginianus) is endemic to properties in the Free State where A. of North America. hebraeum has become established foci of infected ticks may arise with serious consequences. Other species suspected to be susceptible to heart- water but lacking in definitive proof are nilgai (Bose-

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water in domesticated ruminants, and is seen in both non-indigenous and indigenous cattle, sheep and goats. Animals with the acute form of heartwater usually die within a week.

Disease begins with pyrexia, which may exceed 41°C within 1–2 days after onset. It remains high for 4–5 weeks with small fluctuations and drops shortly before death. Fever is followed by inappetence, some- times listlessness, diarrhoea (par- ticularly in cattle), and dyspnoea indicative of lung oedema.

Nervous signs develop gradually, and are generally less pronounced in sheep and goats than cattle. The animal is restless, walks in circles, makes sucking movements and stands rigidly with tremors of the su- perficial muscles. Cattle may push Figure 1 Madder Horak and Stoltsz their heads against a wall or present aggressive or Madder, Horak and Stoltsz illustrate distribution of anxious behaviour. A. hebraeum in South Africa as follows. In the terminal stage the animal falls to the ground Occurrence in lateral recumbency, pedalling and exhibiting Heartwater occurs in nearly all the sub-Saharan opisthotonos, nystagmus, hyperaesthesia, chewing countries of Africa where Amblyomma ticks are pres- movements, and frothing at the mouth. The animal ent and in the surrounding islands: Madagascar, Re- usually dies during or following such an attack. On union, Mauritius, Zanzibar, the Comoros Islands and rare occasions, heartwater occurs as a subacute Sao Tomé. The disease is also reported in the Carib- disease with prolonged fever, coughing and mild in- bean (Guadeloupe, Marie-Galante and Antigua), coordination. CNS signs are inconsistent in this form. from where it threatens the American mainland. The animal either recovers or dies within 1 to 2 weeks.

Symptoms Mild or subclinical infections may be seen in young The average incubation period in natural infections calves, lambs or kids; partially immune livestock; is 2–3 weeks, but can vary from 10 days to 1 month. some indigenous breeds; and some wild ruminants. The incubation period after intravenous blood in- The only sign may be a transient fever. Morbidity is oculation is seven to 10 days in sheep and goats, highly variable and depends on the degree of tick and 10 to 16 days in cattle. However, the incuba- infestation, previous exposure to infected ticks, and tion is strongly dependent on the inoculated dose of level of acaricide protection. elementary bodies. The outcome can range from 100% death with high doses to 0% death with low Once signs of the disease have developed, the doses followed by protection of animals. prognosis is poor for non-native and exotic sheep, goats, and cattle. The mortality rate in non-native Heartwater occurs in four different clinical forms, de- breeds of sheep and goats may be 80% or higher, termined by variations in host susceptibility, agent in contrast to 6% in native breeds. In cattle, mortality virulence and infective dose. Peracute disease is of 60–80% is not uncommon. Recovery from heart- usually seen in Africa in non-indigenous breeds of water infection usually results in complete immunity sheep, cattle and goats. Heavily pregnant cows are against homologous strains, although animals re- particularly susceptible to this form. Peracute dis- main carriers of infection. ease is characterised by sudden death preceded by a brief interval of fever, severe respiratory distress, Pathology and Diagnosis hyperesthesia, lacrimation and, in some breeds of The pathology caused by heartwater is suggestive cattle, severe diarrhoea. Terminal convulsions may but would not allow for a definitive diagnosis. A full be seen. This form of heartwater is relatively rare. set organ samples (brain, lung, heart, liver, spleen, Acute disease is the most common form of heart- kidneys, lymph nodes, gastrointestinal sections and

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muscle) should be collected in 10% formalin to con- Histology of formalin fixed tissues is often unreward- firm the presence of lesions consistent with heartwa- ing but IMP staining on such tissue has been suc- ter. cessfully used to detect the presence of antigen in formalin fixed tissue samples collected from typical The macroscopic lesions observed are fairly similar lesions during post mortem. The brain would be the in cattle, sheep, and goats. The accumulation of organ of choice. fluid (straw coloured to more reddish) is typical and carcasses may present with lesions of hydropericar- Serological tests include indirect fluorescent anti- dium, hydrothorax, pulmonary oedema, oedema of body tests, enzyme-linked immunosorbent assays the mediastinal and bronchial lymph nodes. Con- (ELISAs) and Western blotting. However, cross-reac- gestion may be seen in many organs along with vari- tions with Ehrlichia spp may occur in some these able degrees of petechiation of organs and serosal tests. These tests have specific application and may surfaces. be employed to monitor experimental infections, to check the immune status of immunised animals, Petechiae on the epicardium and endocardium and to screen animals prior to importation. As diag- and congestion of the brain may be prominent. Par- nostic test it is of less value. enchymatous organs may be enlarged and mod- erate splenomegaly may be seen. Pulmonary oe- The MAP1-B ELISA and the MAP1 competitive ELISA dema may be prominent and froth in the trachea is have been shown to have a higher specificity for E. often seen. Macroscopic lesions are not usually seen ruminantium, and it does not cross-react with anti- absent except for subtle swelling of the brain, which bodies against Anaplasma bovis, Anaplasma ovina may result in herniation through the foreman mag- and Anaplasma phagocytophila. The MAP 1B ELISA num. Congestion and/or oedema of the abomasal test does, however, detect antibodies to other Eh- folds are commonly observed in cattle, but less so in rlichia spp including E. canis, E. chaffeensis, an un- sheep and goats. Nephritis of varying degree, espe- identified Ehrlichia sp, infecting white-tailed in the cially in Angora goats, may be seen. south-eastern USA, and E. ruminantium (Omatjenne). The MAP 1B ELISA test works well in sheep and goats, A diagnosis of heartwater may be confirmed follow- but not cattle. In cattle antibody levels against E. ru- ing the demonstration of colonies of E. ruminantium minantium can be very low in heartwater endemic in brain smears. The entire brain should preferably areas, even in vaccinated cattle or continuously be removed and the hippocampus is the preferred naturalle challenged by infected ticks. Cattle may region to sample but the cerebral grey matter could become seronegative 14 to 33 weeks after initial ex- also be collected. posure. A serum sample, in a red or yellow stopper tube, is required. It is to be stored at 4° C and trans- Brain tissues are crushed and smears are prepared ported to the laboratory as soon as possible on ice. spreading the crushed brain material thinly and more thickly, at regular intervals, in a single cell lay- PCR assays generally are less suitable for large-scale ers. Smears are the air-dried, fixed with methanol testing and therefore not likely to replace serologi- and stained with Giemsa or Diff Quick to demon- cal tests in epidemiological studies. PCR assays are strate the presence organisms in the capillary endo- useful as confirmatory tests and in some specific in- thelial cells of the brain. Smears can be fixed and stances such as regulatory testing, research on the stained in less than one minute. Heartwater colonies Ehrlichia genome and for epidemiological studies. are reddish-purple to blue, and seen very often Blood samples, in EDTA tubes are suitable. PCR can close to the nucleus of the infected endothelial cell. also be attempted on other target organs, such as More than one smear should routinely be prepared brain, lungs, kidneys, and thoracic fluids Samples as organisms may be very few and difficult to find, are to be stored at 4° C and transported to the labo- particularly in peracute cases. However, they should ratory on ice, as quickly as possible. always be present in the brain of an animal that has succumbed to heartwater. E. ruminantium may be isolated from the blood of an infected host by cultivation on ruminant endothe- Colonies may still be visible for a period of two days lial cells and the presence of characteristic morulae after death in brains stored at tolerable room tem- may be confirmed by immunofluorescence or im- perature; and up to 34 days in brains stored in a re- munoperoxidase techniques using a specific anti- frigerator. Organisms could also still be seen in the serum. Blood samples, in EDTA tubes are suitable. brains of animals with advanced autolysis. Fixed or Samples are to be stored at 4° C and transported to unfixed smear may still suitable for a diagnosis for at the laboratory on ice, as quickly as possible. least one month after having been prepared. Dif- ficulties may be experienced in demonstrating or- Differential Diagnosis ganisms if he animal has been treated with drugs (48 The peracute form of heartwater may be confused – 60 hours previously) or in peracute clinical cases. with anthrax. While the acute form may resemble

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rabies, tetanus, bacterial meningitis or encephalitis, crumbles per Kg of feed for 25 days. A control group babesiosis, anaplasmosis, cerebral trypanosomia- of ten sheep had no antibiotic added to the feed. sis, or theileriosis. It must also be differentiated from On the second day of feeding he inoculated all the poisoning with strychnine, lead, ionophores and sheep with 5ml of fresh sheep's blood infected with other myocardial toxins, organophosphates, arse- the Ball 3 strain of heartwater after which tempera- nic, chlorinated hydrocarbons, or some poisonous tures were taken daily. The sheep were observed for plants. Accumulations of fluid similar to heartwater a period of 38 days. All surviving sheep were chal- are also sometimes seen in heavy helminth infesta- lenged on the 38th day by the intravenous inocula- tions (haemonchosis). tion of 5 ml of Ball 3 heartwater blood. Eleven sus- ceptible sheep were also inoculated as controls. Treatment and Control Only one of the thirty antibiotic fed sheep showed P G Marais reports that several drugs have been typical heartwater during the 38-day period post used with varying success to treat animals suffer- inoculation. All the remaining sheep were immune ing from heartwater. Tetracyclines, especially oxy- when challenged with heartwater after 38 days. tetracycline, however, are most effective and are widely used for the specific treatment of the disease. All ten untreated control animals developed typi- Short-acting formulations of oxytetracycline are most cal heartwater, with febrile responses of up to 42°C, commonly used at a dosage rate of 10-20 mg/kg hyperaesthesia, rapid breathing, and finally pros- body weight, either administered intramuscularly as tration, galloping movements and foaming at the a single dose, or half the calculated dose is given mouth and nose. Despite efforts to salvage these intravenously and the other half intramuscularly. This sheep by the parenteral administration of antibiot- treatment is usually repeated 24 hours later. A long- ics, 7 out of 10 died. Dead sheep were autopsied, acting oxytetracycline preparation has given similar and brain smears prepared, stained with Giemsa, results to the short- acting formulations of the drug in and examined for the causal agent of heartwater. the treatment of clinical cases of heartwater. Vaccination currently consists of infection with a live OIE recommendations include oxytetracycline at 10 E. ruminantium strain, then treatment with antibiot- mg/kg or doxycycline at 2 mg/kg during the early, ics when a fever develops. Alternatively, the vaccine febrile stages of this disease, but they suggest that may be given to young kids or lambs during their animals often die before treatment can be admin- first week of life, or to calves less than 5 to 8 weeks istered and antibiotic treatment alone is not always of age; young animals possess a degree of non- successful in later stages. Tick control is imperative specific resistance to infection, and do not always because E. ruminantium cannot survive outside a require treatment. Vaccination does not always pro- living host for more than a few hours at room tem- tect animals from all field strains, and revaccination perature and heartwater is usually introduced into is risky due to the possibility of anaphylactic reac- free areas by infected animals, including subclinical tions. This method is likely to be replaced with inacti- carriers, or by ticks. vated and attenuated vaccines in the future.

Carrier animals may be detected by serological A first-generation vaccine consisting of inactivated tests before introduction, but these tests have limi- purified elementary bodies of E. ruminantium emul- tations according to the OIE. Animals moved into sified in Montanide ISA 50 adjuvant has given good endemic areas may be protected by chemopro- results in experimentally controlled conditions and phylaxis. PG Marais describes this as a procedure by has demonstrated significant protection in the field. which a series of oxytetracycline injections is used to protect susceptible animals against heartwater Three different isolates (Senegal, Gardel and Wel- when they are introduced into an endemic area. He gevonden) have been attenuated and shown to suggests that the development of such a prophylac- confer good protection, and significant protection tic regime holds great promise as a practical and has also been obtained using DNA vaccination. reasonably safe method for the introduction of large However, none of these new experimental vaccines groups of animals into endemic areas. However, has been fully validated under field conditions. Field he says, its success is almost exclusively dependent trials have revealed that antigenic diversity is impor- on all the animals becoming naturally infected with tant in formulating effective vaccines, and further heartwater during the time that they are protected investigations are critical for the delivery of any vac- by the oxytetracycline. cine in the field.

During 1972 CJ Mare reported investigating pro- Inactivated Vaccines longed oral administration of oxytetracycline to Inactivated vaccines using the Gardel strain and manage artificially-induced heartwater in sheep. He subsequently other strains have been produced in fed 10 sheep per group 500g per day of meal con- bioreactors for an industrial production following the taining 0.2, 0.3 or 0.4 kg of Terramycin A/D fortified development of the whole production process. The

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vaccine produced has the same efficacy as the ini- The main drawback of attenuated vaccines is their tial laboratory produced experimental vaccine and extreme lability, which necessitates their storage in a production cost calculated at around 0.1 € per liquid nitrogen and their distribution in frozen condi- dose which becomes acceptable. The Mbizi strain tions. In addition, they have to be administered in- inactivated vaccine is being developed commer- travenously. cially by Onderstepoort Biological Products in South Africa. Recombinant Vaccines Several reports show partial protection of mice using These inactivated vaccines do not prevent infec- map1 DNA vaccination and an improvement of pro- tion but do prevent or reduce death of vaccinated tection by vaccination following a prime (plasmid) animals when exposed to live virulent challenge. – boost (recombinant MAP1) protocol. However, pro- They confer protection for more than one year. The tection of ruminants has never been demonstrated advantage is that several field strains can be incor- using this strategy. porated to make the vaccine more widely cross- protective. In opposition, significant protection of sheep was reported against homologous and heterologous ex- A major challenge remains the characterisation of perimental challenge following plasmid vaccination the extent of strain diversity in a region to be covered using a cocktail of four ORFs (open reading frames) by an appropriate formulation of the vaccine. This from the 1H12 locus in the E. ruminantium genome. knowledge will also be essential for new generation No further results have been described since then. vaccines that will be developed in the future. Recombinant vaccines will probably not be avail- Live Attenuated Vaccines able soon. Infection of ruminants with live E. ruminantium strains induces a strong long-lasting protection against an Conclusion homologous isolate. This is the basis for infection and Veterinarians will need to be alert to the spread of treatment using virulent isolates. Isolates of attenu- this disease into previously uninfected areas as en- ated virulence that do not necessitate treatment vironmental conditions change and free movement of animals would be ideal, but a limited number of of game species and domestic ruminants occurs such attenuated isolates are available. References

1. OIE HEARTWATER – COWDRIOSIS Aetiology Epidemiology Diagnosis Prevention and Con- An attenuated Senegal isolate has been obtained trol References and shown to confer 100% protection against an 2. Marais PG, Lets talk Heartwater (Cowdria ruminantium) Grootfontein Agricultural Devel- homologous lethal challenge, but very poor protec- opment Institute - http://gadi.agric.za/articles/Agric/artikel_hartwater.php tion against a heterologous challenge. The Gardel 3. Mare CJ, The Effect of Prolonged Oral Administration of Oxytetracycline on the Course isolate, which gives a significant level of cross-pro- of Heartwater (Cowdria ruminantum) Infection in Sheep. Trop. Anita. Hlth Prod. (1972) 4, tection with several isolates (although far from com- 69-73 plete), has also been attenuated. 4. Horak, I.G., Jordaan, A.J., Nel, P.J., Van Heerden, J., Heyne, H. & Van Dalen, E.M., 2015,

‘Distribution of endemic and introduced tick species in Free State Province, South Africa’, Recently, a third isolate named Welgevonden from Journal of the South African Veterinary Association 86(1), Art. #1255, 9pages. http:// South Africa has been attenuated and shown to dx.doi.org/10.4102/jsava.v86i1.1255 confer complete protection against four heterolo- 5. Madder M, Horak IG, Stoltsz H. Tick identification http://www.itg.be/photodatabase/Afri- gous isolates under experimental conditions. can_ticks_files/index.html

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