Life Cycle of Potomac Horse Fever - Implications for Diagnosis, Treatment, and Control: A Review

Neorickettsia risticii is the agent of the disease often called Potomac Horse Fever (PHF) that can manifest as colic, diarrhea, enterocolitis, or abortion in pregnant mares. The agent is found in nature in a complex life cycle involving flukes, freshwater snails, and aquatic insects that are consumed by bats and birds. Horses become infected when consuming the infected adult insects, such as caddisflies, which harbor N. risticii in a metacercariae phase of a fluke. Diagnosis is based on clinical signs and available real time TaqMan polymerase chain reaction (PCR) on blood and/or feces.

1. Introduction

Neorickettsia risticii [1] was isolated from the blood of a horse with clinical signs of colic and diarrhea nearly 20 years ago. Only recently has the agent been found in nature (in freshwater snails and aquatic insects), the life cycle been determined, and the mode of transmission been experimentally reproduced. The purpose of this paper is to review recent developments in the determination of the life cycle of N. risticii and describe diagnostic methods, treatment, and control measures for Potomac Horse Fever (PHF).

2. Infectious Agent

Horses with clinical symptoms of fever, colic, or diarrhea followed by laminitis was first recognized as being a distinct syndrome by veterinarians along the Potomac River in Maryland [2]. Blood transmission from an affected clinical case to a control horse reproduced the syndrome and eventually lead to isolation of a rickettsial agent initially called Ehrlichia risticii and now renamed Neorickettsia risticii. N. risticii is the etiologic agent of PHF, also called equine monocytic ehrlichiosis, equine ehrlichial colitis, or Shasta River Crud. Genetic analysis has recently lead to reclassification based on the relation between three other species - N. sennetsu (human agent of Sennetsu fever), N. helminthoeca (agent of salmon poisoning in the dog), and Stellantchasmus falcatus (SF agent; an ehrlichia-like bacterium present in the metacercarial stage of the fluke) [1]. Strain variance has been determined between 11 N. risticii strains with a maximum divergence of 0.7%. N. risticii is a gram-negative coccus and stains dark blue to purple with Giemsa stain and Romanowsky's stain; however, it cannot be visualized on blood films in monocytes or other cells. The agent can be grown in cell culture but cannot be isolated in conventional bacterial culture systems. N. risticii has been successfully grown in human histiocytic lymphoma cells as well as canine, equine, and murine monocytes.

3. Epidemiology

After isolation of N. risticii from the blood of horses along the Potomac River in Maryland, horses with similar clinical signs were believed to be suffering from PHF. Serological surveys of horses were conducted to determine antibodies to N. risticii. Based on the indirect fluorescence antibody test (IFA), PHF has been suggested to occur in 43 states in the United States, three provinces (Nova Scotia, Ontario, and Alberta) in Canada, South America (Uruguay and Brazil), Europe (The Netherlands and France), and India. Determining the true extent of the disease in the United States and elsewhere has been hampered by the use of the IFA test for antibodies to the causative agent, because the test produces >30% false positives [3]. Isolation or detection of the causative agent from clinical cases of the disease using conventional cell culture or the polymerase chain reaction (PCR) assay has only been reported in 14 states (California, Illinois, Indiana, Kentucky, Maryland, Montana, Michigan, New York, New Jersey, Ohio, Oregon, Pennsylvania, Texas, and Virginia), Nova Scotia, Uruguay, and Brazil.

Defining the epidemiology of N. risticii has been the subject of intensive research efforts for over 20 years. Initially, the disease was classified as an Ehrlichia, and because all members of that species were believed to be tick transmitted, a long unrewarding hunt for the vector of PHF took place over the last 20 years [4]. Recent evidence on the DNA relatedness of the agent to members in the Neorickettsia group by researchers at Ohio State provided new basic information on the agent [5]. This relatedness included the agent of Salmon poisoning in dogs (N. helminthoeca). Because PHF commonly occurs near freshwater streams and rivers and on irrigated pastures (usually during mid to late summer [May to November]), our research group began to undertake an investigation into the role of freshwater snails in the epidemiology of PHF. We developed reliable PCR tests for the detection of N. risticii DNA in materials from aquatic environments. Using an area in northern California that was endemic for a syndrome historically termed Shasta River Crud, which we previously determined was caused by N. risticii, we collected freshwater snails from the Shasta River. We found DNA evidence of N. risticii in operculate snails (Pleuroceridae: Juga spp.) of N. risticii. Furthermore, we determined that the snails contained trematodes, which also tested positive for N. risticii DNA [6,7]. These operculate freshwater snails act as intermediate hosts for fluke stages, and we considered that they may be involved in the life cycle of N. risticii. The results of sequencing PCR-amplified DNA from a suite of genes (16S rRNA, groESL heat shock operon, and 51-kDa major antigen genes) indicated that the source organism was clearly related to the type strain of N. risticii [7]. The number of snails harboring the trematode stages varied from 3.3% to 93.3%, and the number of PCR-positive snails (3.3-20%) varied with the size of the snails, the month of collection, and the geographic origin. In northern California, the species of snail in the life cycle of N. risticii has been identified as Juga yrekaensis, a common pleurocerid snail that inhabits fresh or brackish stream water in the northwestern United States. Additionally, DNA from N. risticii has been detected in virgulate cercariae in lymnaeid snails (Stagnicola spp.) from northern California, in virgulate xiphidiocercariae isolated from pleurocerid snails (Elimia livescens) in central Ohio, and from pleurocerid snails (Elimia virginica) in central Pennsylvania, suggesting that other types of snails may also harbor infected trematodes [8,9]. This type of trematode is known to become encysted in the second intermediate host, and N. risticii DNA has been detected in mesocercaria and metacercaria in various aquatic larval, nymphal, and adult insects such as caddisflies, mayflies, damselflies, and dragonflies initially found in northern California and subsequently detected in central Pennsylvania [9,10]. Recently, we identified two potential helminth vectors (Acanthatrium spp. and Lecithodendrium spp.), which were both infected with N. risticii, in the intestine of bats and birds collected in northern California [11]. This finding, which was duplicated by the group headed by Rikihisa using species found in Pennsylvania, was published by Gibson et al [12]. These trematodes belong to the Lecithodendriidae family, which are common parasites of bats, birds, and amphibians in North America that use pleurocerid freshwater snails as first intermediate hosts and aquatic insects as second intermediate hosts. Further studies may reveal additional trematodes or members of the Lecithodendriidae or other families that may also act as vectors of N. risticii in other endemic regions of the United States.

The definitive host for N. risticii remains questionable. Antibody titers to N. risticii have been found in domestic and wild animals such as dogs, cats, coyotes, pigs, and goats from regions in which PHF is endemic [13]. Additionally, a variety of non-equine mammalian species, such as mice, dogs, cats, and cattle, have been shown to be susceptible to N. risticii [14-16]. Based on N. risticii DNA sequence detection in the blood, livers, and spleens of bats and swallows, we speculate that these insectivores may act as both a definitive host of the helminth vector and a natural reservoir of N. risticii [11,12].

4. Transmission

Given the complex life cycle of this agent, the means of natural transmission was investigated in several ways. The proof that the DNA evidence found in nature was correlated with the clinical disease manifestations of PHF in horses was studied by inoculating the PCR-positive trematode stages into horses and mice. Horses that were injected subcutaneously with N. risticii PCR-positive trematode stages (virgulate cercariae and sporocysts) collected from J. yrekaensis snails developed clinical signs of colic and diarrhea and hematological changes consistent with PHF [17]. Furthermore, N. risticii was transmitted to mice using PCR-positive metacercariae isolated from caddisfly larvae (Dicosmoecus spp.) [10]. The final proof of insect ingestion transmission came from a collection of caddisfly larvae from the endemic areas for PHF that were hatched into mature flies and placed in the feed of an experimental horse. Ten days later, the horse developed the full spectrum of clinical signs of PHF and related hematological changes. Cell culture isolation, PCR detection, gene sequence identification, and reinoculation of the isolate into additional horses produced the same syndrome, fulfilling Kock's postulates [18]. This work was repeated by the Ohio State group using caddisfly species collected from non-California regions, which again confirmed the natural transmission by oral ingestion of insects harboring N. risticii in metacercariae [9]. Therefore, aquatic insects, such as caddisflies and mayflies, represent a likely source of infection because of their abundance in the natural environment, their high infection rate with N. risticii determined by PCR, and the mass hatches regularly observed during summer and fall. Under natural conditions, horses grazing near rivers or creeks will ingest adult insects along with grass (adults live near water and are likely to die there), consume adult insects trapped on the water surface, or possibly, consume adult insects that, attracted by stable lights, accumulate in feed and water.

5. Clinical Findings

Naturally occurring cases of PHF are typified initially by an acute onset of mild depression and anorexia, followed by a biphasic increase in body temperature ranging from 38.9°C to 41.7°C (102-107°F) [19]. At this stage, decreased intestinal sounds are often noted. Within 24 - 48 h, a moderate to severe diarrhea ranging from cowpie to watery consistency develops in ~60% of affected horses. The onset of diarrhea is often accompanied by mild colic signs. Some horses develop severe toxemia and dehydration; this results in cardiovascular compromise that is characterized by elevated heart and respiratory rates and congested mucous membranes. Subcutaneous edema along the ventral abdomen may be observed. Laminitis is a sequela in many cases. The associated laminitis may progress, despite resolution of other clinical signs. Interestingly, laminitis has only been reported in naturally infected horses, which probably reflects undetermined pathophysiological mechanisms related to the natural route of transmission. It should be emphasized that a case of PHF may present with all or any combination of the aforementioned clinical signs. Case fatality rates vary from 5% to 30% and may depend on the strain involved and host response. Long-term problems seem to be related to sequelae such as laminitis. To date, no evidence exists that N. ristici infection results in chronic disease, and many attempts to isolate N. riscii by culture or PCR after clinical signs have abated have been unsuccessful.

Transplacental transmission of N. risticii can occur and has been seen in natural and experimental infections [20,21]. The organism may induce abortion, cause resorption of the fetus, or produce weak foals. There are reports of pregnant mares developing clinical signs of PHF and subsequently, aborting at ~7 mo gestation, regardless of the severity of infection [21]. In mares experimentally infected at 90-120 days of gestation, abortion occurred at 65-111 days after inoculation [21]. Abortions are spontaneous with a fetus appearing in fresh condition.

Hematological findings vary in the early stage of PHF and are not useful in making a definitive diagnosis. Findings vary from a transient leucopenia (a white blood cell count of <5000/μl), which is characterized by a neutropenia and a lymphopenia, to a normal hemogram, even with evidence of systemic toxicity. [22] Other cases of PHF develop leukocytosis (>14,000/μl), which is normally observed within a few days of onset of the disease. Increase in both packed cell volume and plasma protein concentration secondary to dehydration and hemoconcentration can occur. A transient non-regenerative anemia and thrombocytopenia may develop.

6. Diagnosis

Serology

A provisional diagnosis of PHF is often based on the presence of clinical signs and the seasonal and geographical occurrence of the disease in an endemic area. A definitive diagnosis of PHF, however, should be based on the isolation or detection of N. risticii from the blood or the feces of infected horses. Serological tests using indirect fluorescent antibodies or enzyme-linked immunosorbent assay tests are of limited value as a diagnostic tool in a clinical case. Antibody levels to N. risticii have unpredictable patterns in individual horses at the time of onset of clinical disease. Antibodies may not reach detectable levels for some period of time after infection, or conversely, long incubations may allow the titer to peak at the time of initial presentation. Paired serum tiers may not show four-fold rises, because the titer may be elevated at the time of initial presentation. Elevated single titers have been used for diagnosis; however, this test should be considered unreliable, because elevated single titers may overlap with levels found in healthy horses with no evidence of the disease. Additionally, the reliability of the indirect immunofluorescence technique for antibody detection has been questioned, because the test yields a high percentage of false-positive results [3]. Isolation of the agent in cell culture from the peripheral blood of affected patients, though possible, can take from several days to several weeks of culturing before detection is successful. Furthermore, it is not routinely available in many diagnostic laboratories.

PCR

The recent development of N. risticii-specific PCR assays has greatly facilitated the diagnosis of PHF [23,24]. In experimentally and naturally infected animals, PCR performed on feces and peripheral blood was more sensitive than cultures [25]. Conventional PCR assays are, however, time consuming and prone to contamination. Novel real-time-PCR platforms associated with automated nucleic acid extraction allow the detection of N. risticii DNA within the day, making this technology a more feasible test for routine diagnostic examination [26]. To enhance the chances of detection of N. risticii, the assay should be performed on blood as well as on a fecal sample, because the presence of the organism in blood and feces may not necessarily coincide. PCR is also used in the detection of N. risticii DNA in fresh or formalin-fixed and paraffin-embedded colon tissue, which aids post-mortem diagnosis.

Differential diagnosis should include all causes of enterocolitis, such as salmonellosis, clostridial diarrhea, intestinal ileus secondary to displacement or obstruction, and peritonitis. Diagnostic tests specific to ruling out these diseases should be concurrently pursued.

7. Treatment

Prompt treatment of PHF cases with oxytetracycline (7 mg/kg, q 12 h, IV) can reduce the morbidity and mortality of the disease. Supportive treatment as needed for enterocolitis cases is indicated. Dehydration occurs rapidly, and fluid and electrolyte balance must be maintained. Prevention strategies for laminitis are indicated. Delay in treatment with oxytetracycline may lessen the therapeutic value of the antibiotic.

8. Prevention

In light of recent epidemiological discoveries concerning the vector of N. risticii and its helminth hosts, horses could conceivably be exposed to N. risticii through skin penetration by infected cercariae or by consuming infected cercariae in water, or metacercariae in a second intermediate host such as an aquatic insect. One horse fed adult caddisflies (Dicosmoecus gilvipes) in northern California [18] and two horses fed adult caddisflies (Cheumatopsyche campyla, Hydropsyche hageni) or a mixture of adult caddisflies and mayflies (Leucrocuta minerva) in central Pennsylvania developed PHF [9]. These studies attempted to mimic the natural route of infection with N. risticii and show that oral transmission using infected aquatic insects was not only possible but also that the clinical disease produced was similar to that seen in naturally infected horses. Aquatic insects, such as caddisflies and mayflies, represent a likely source of infection because of their abundance in the natural environment, their high infection rate with N. risticii determined by PCR, and their mass hatches that are regularly observed during summer and fall. Under natural conditions, horses grazing near rivers and creeks will consume adult insects along with grass (adults live near water and so are likely to die there), ingest adult insects trapped on the water surface, or possibly, ingest adult insects that are attracted by stable lights and subsequently accumulate in feed and water.

Does vaccination protect against naturally occurring PHF? The answer to this question is unknown, because horses have only been challenged after immunization with the available IV vaccines (not oral vaccines). Additionally, the presence of antibodies does not always correlate with the clearance of Neorickettsial organisms and the presence of protective immunity. This has been shown with horses that have been vaccinated with a killed N. risticii vaccine and subsequently, developed the clinical disease after natural exposure [27]. Antibodies induced by a killed vaccine may not be effective, because protective antigens may only be expressed during cell invasion or replication. It is likely that cell-mediated immunity plays a dominant role in protecting the host from N. risticii infection as shown in other rickettsial infections.

In local environments where PHF is a problem, methods to reduce snail numbers in adjacent creeks, ditches, or other bodies of water should be considered. If possible, water should be drained from ditches.