Neorickettsia helminthoeca

Salmon poisoning disease was first recognized in the early nineteenth century by settlers in the Pacific Northwest when their dogs fell ill after consuming raw salmon. The causative agent, N. helminthoeca, was characterized in 1953, but could not be placed in a previously described genus based on the disease and morphologic characteristics. It is unique, in that it is the only obligately helminth-borne pathogenic bacterium. Canine mononuclear cells are infected after dogs ingest salmonid fish encysted with a fluke, Nanophyetus salmincola (Figure 40-1), infected with the organism. The disease is indigenous to river areas of the U.S. Pacific Northwest coast, from northern California to southwestern Washington State. Occasional cases associated with fish migration have been reported in British Columbia.

FIGURE 40-1 Life cycle of Nanophyetus salmincola.

(Courtesy Ashley E. Harmon.)

The raccoon and spotted skunk are the principal definitive hosts for the fluke. Adult flukes develop in the intestinal lumen within 6 days after ingestion in dogs, cats, foxes, bears, coyotes, opossums, otter, mink, lynx, and birds. Infected eggs shed in feces hatch and release a miracidium. The miracidium penetrates the first intermediatehost, the snail Oxytrema silicula, where the cecariae (free-living trematode larvae) develop. Cercariae are liberated and penetrate the skin of the secondary intermediate host, which are fish, usually of the family Salmonidae, or Pacific giant salamanders. Metacercariae are found in the muscle, kidney, eye, and other organs of fish and salamanders. Neorickettsia helminthoeca is maintained by transovarial passage in the helminth and is found throughout the life cycle of the fluke, including free-swimming cercariae.

Salmon poisoning (Figure 40-2) generally affects only members of the family Canidae, but death of captive polar bears has been reported in association with salmon poisoning disease. After ingestion of infested fish, the trematodes attach to and penetrate deeply into the mucosa, particularly in the duodenum but also throughout the small and large intestines. The precise mechanism of infection remains to be elucidated, but superficial enteritis develops rapidly, and progresses to hemorrhagic enteritis. Bacteria spread by the hematogenous route to lymph nodes, spleen, liver, lungs, brain, and thymus. Acute disease is characterized by fever, depression, dehydration, anorexia, diarrhea, and lymphadenopathy. The case fatality rate in untreated dogs is 50% to 90% and the exact cause of death remains unknown. Recovered animals are immune to reinfection.

FIGURE 40-2 Lymph node with lesions of salmon poisoning, showing accumulation of bacteria-laden macrophages in the subcapsular sinus and amongthe medullary cords. Edema is evident.

(Courtesy Raymond E. Reed.)

Difficulties related to bacteriologic culture of N. helminthoeca have been a detriment to studying disease pathogenesis. Recently propagation in a continuous cell line has facilitated antigenic and genetic analyses. Canine monocytes, canine leukocytes, mouse lymphoblasts, and a macrophage line support in vitro growth.

Antemortem diagnosis is based on detection of fluke eggs in feces, presence of compatible clinical signs, history of travel to the Pacific Northwest, demonstration of rickettsiae in lymph node aspirates, and serology. Trematode eggs are operculated and are passed in the feces 5 to 8 days after fish ingestion. They can be detected by direct smears or sugar flotation techniques. Macchiavello or Giemsa stains of lymph node aspirates reveal intracytoplasmic rickettsial bodies, but organisms are not detected by microscopic examination of circulating lymphocytes. Affected animals typically seroconvert 13 to 15 days postinfection, so several veterinary diagnostic laboratories offer serologic diagnosis by way of indirect fluorescent antibody or complement fixation tests.

Postmortem diagnosis is based on compatible gross and microscopic lesions. Changes areprimarily associated with lymphoid tissues, and include lymphadenopathy, splenomegaly, and petechiation of the gallbladder, pancreas, and mucosae of the esophagus, urinary bladder, and intestinal tract. General lymph node enlargement is attributed to marked infiltration of macrophages. Microscopic lesions include depletion of mature lymphocytes from lymph node cortex and medulla, nonsuppurative meningoencephalitis, splenic follicular central hemorrhage and necrosis, and obliteration of thymic architecture. Coccoid bodies may be found within the macrophages.

Prevention of the disease involves not allowing raw or improperly cooked fish to be fed to dogs. Thorough cooking, or freezing at −20° C for24 hours, kills N. helminthoeca and metacercariae. If infected raw fish is eaten, apomorphine should be administered as an emetic. Sick animals are given supportive care to control vomiting and diarrhea, and to maintain acid-base balance. Parenteral administration of tetracyclines is usually helpful, but there are no commercial vaccines.

Elokomin Fluke Fever

Elokomin fluke fever (EFF), which is similar to salmon poisoning disease, was first described in the Elokomin River valley of Washington State. The etiologic agent is suspected to be a less virulent strain of N. helminthoeca. The organism has been referred to as “Neorickettsia elokominica,” and EFF is also associated with ingestion of raw fish. Clinical features distinguishing this disease from salmon poisoning are incubation period, character of the febrile response, persistence of lymphadenopathy, low mortality, and lack of conferred immunity between the two diseases. The incubation period for EFF, at 5 to 12 days, is slightly longer than that for salmon poisoning. The period of elevated temperature is extended, lasting 4 to 7 days. Other clinical signs are identical to, but less severe than, those observed in salmon poisoning. Bears, in addition to dogs, appear to be susceptible to EFF. Serologic differentiation between EFF and salmon poisoning disease has been accomplished by way of the complement fixation test.

Neorickettsia (Ehrlichia) risticii

Neorickettsia risticii is the agent of Potomac horse fever (PHF), an acute diarrheic illness of equids, also known as equine monocytic ehrlichiosis or equine scours, because of the affinity of the organism for blood monocytes, tissue macrophages, and intestinal epithelial cells. The disease was first recognized in 1979 in areas bordering the Potomac River in Virginia and Maryland. Since then it has been diagnosed in most states within the continental United States, several Canadian provinces, and South America (Brazil, Venezuela, and Uruguay). Serologic evidence of PHF has been found in France, India, and Australia.

The epidemiology of PHF has remained obscure since its discovery more than 20 years ago. The disease appears to be restricted to low-lying regions in proximity to bodies of water, and there is a seasonal pattern with peak incidence occurring during summer. Arthropod vectors do not appear to be involved in transmission, but N. risticii is found in the feces of infected horses. Oral transmission has been demonstrated experimentally, but this route is not natural.

Neorickettsia risticii appears to be maintained in nature in a complex aquatic ecosystem. The infection cycle apparently involves an intermediate snail reservoir and a trematode cercarial vector. In the laboratory, researchers have infected mice and horses by intraperitoneal subcutaneous inoculation, respectively, with trematodes from snails. DNA of N. risticii has been found in virgulatecercariae from the freshwater snails Juga yrekaensis in northern California and Elimia livescens in Ohio. Such DNA has also been detected in metacercariae from aquatic insects, such as caddisflies, stoneflies, damselflies, mayflies, and dragonflies. Oral transmission of PHF has been demonstrated in horses fed caddis flies, and it is natural to speculate that transmission to horses involves accidental ingestion of insects harboring infected metacercariae. Potential helminth vectors include Lecithodendrium and Acanthatrium spp. These N. risticii–infected helminths have been found in the intestinal tracts of bats and birds, but no definitive reservoir host has been identified. Besides horses, other susceptible mam-mals include cattle, mice, dogs, and cats. In endemic locations, antibody titers to N. risticii have been found in goats, pigs, cats, dogs, and coyotes.

The primary clinical sign is acute, waterydiarrhea. Mild colic, anorexia, fever, depression, edema, dehydration, laminitis, and leukopenia are additional findings. Rarely, abortion may result from infection in the unborn fetus. The case fatality rate is 5% to 30%. Fatalities result if severely affected horses are not treated promptly with electrolytes, fluids, and appropriate antimicrobial agents.

The watery diarrhea is a direct result of enterocyte infection. Degeneration of infected epithelial cells, with loss of microvilli, results in accumulation of cAMP and physical inability to conduct electrolyte transport in the colonic mucosa. Malabsorption of sodium and chloride ions and lack of water resorption lead to diarrhea.

A diagnosis of PHF is suggested in horses exhibiting nonspecific signs and diarrhea during the summer months in areas endemic for N. risticii. Several options are available for the diagnosis of PHF, including an indirect fluorescent antibody test. Rising titers with accompanying clinical signs suggests active infection. Detection of the organism in infected tissues has been accomplished using a modified Steiner silver stain, an immunoperoxidase method, or transmission electron microscopy. Definitive diagnosis of PHF formerly required isolation of N. risticii in cell culture, but polymerase chain reaction (PCR) amplification of DNA from peripheral blood or feces has proven to be a highly specific and sensitive means of diagnosis.

Commercial, inactivated vaccines for PHF are available but the antibody response to vaccination has been poor and there are consistent reports of vaccine failure in the field, particularly in endemic areas. Heterogeneous strains of N. risticii have been recovered from field-vaccinated horses suffering from clinical PHF. Thus it appears that both a deficiency in antibody response and heterogeneity among isolates are responsible for the present vaccine failures. Infections can be treated by intravenous administration of oxytetracycline early in the course of the disease.

Neorickettsia (Ehrlichia) sennetsu

The first neorickettsial pathogen of humans was identified in Japan in 1953. Sennetsu fever, caused by N. (Ehrlichia) sennetsu, is characterized by fever and swollen lymph nodes. The disease is very rare outside the Far East and Southeast Asia, with the majority of cases having been reported from western Japan. Epidemiologic studies suggest that infection is acquired through ingestion of raw gray mullet fish infested with the metacercarial stage of trematodes infected with N. sennetsu. This organism is infectious but nonpathogenic for horses, and inoculation with N. sennetsu has protected horses from PHF upon challenge with N. risticii. Laboratory mice are highly susceptible to infection.

Anaplasma (Ehrlichia) bovis

A. bovis infects mononuclear cells of cattle and possibly sheep in tropical and subtropical regions of the world. Disease is characterized by anemia and weight loss, and, rarely, by abortion and death. Survivors are lifelong carriers. Organisms are infrequently observed within the cytoplasmof monocytes in peripheral blood smears. Tick vectors include Amblyomma cajennense, Amblyomma variegatum, Rhipicephalus appendiculatus, and Hyalomma excavatum. Serologic cross-reactions with Ehrlichia ruminantium have been documented.

Anaplasma caudatum

Bovine anaplasmosis in the United States maybe caused by the intraerythrocytic bacteria A. caudatum and A. marginale. Organisms are transmitted biologically by ixodid ticks and mechanically by biting flies or veterinary procedures. Clinical and pathologic signs include anemia, icterus, splenomegaly, gallbladder obstruction, erythrophagocytosis, and hemosiderosis. Pregnant cows may abort. The natural history of infection is characterized by four sequential stages; an incubation period of 3 to 5 weeks isfollowed by patency, with clinical signs lasting2 or more weeks, convalescence of 4 to 8 weeks, marked by increased hematopoiesis and recrudescence, and an indefinitely long carrier state during which hematologic values return to normal. Tetracyclines are used for elimination of thecarrier state.

Anaplasma centrale

Anaplasma centrale produces a natural, persistent infection of bovine erythrocytes. As implied by the name, the bacteria are usually found in the center of red blood cells. Subclinical disease is common, but in some instances fever and anemia without icterus may result. The agent has been employed as an immunizing agent against A. marginale in endemic areas.

Anaplasma marginale

Initially described by Theiler in 1908 as a developmental stage of the protozoan Babesia bigemina, A. marginale is the primary cause of bovine anaplasmosis, an economically significant disease on six continents. Biologic transmission occurs when infected hard ticks in the genera Boophilus, Dermacentor, Ixodes, or Rhipicephalus feed on immunologically naive cattle. Mechanical transmission occurs through biting flies or blood-contaminated fomites such as used hypodermic needles or dehorning instruments. Cattle that recover from acute infection remain persistently infected for years, with microscopically undetectable levels of the organism. Carriers develop clinical disease when stressed. Persistence in primary hosts is fundamental to continued transmission because transovarial transmission of A. marginale in the tick vector does not occur. Anaplasmosis is enzootic in the southern Atlantic and Gulf Coast states, on the lower plains, andin western states, but occurs sporadically in northern states. Natural and biologic vectors are seasonal, so there is a correlation between disease outbreaks and vector seasons.

Anaplasma marginale causes fever, anorexia, weight loss, decreased fertility in bulls, and icterus. Acute disease is characterized by severe anemia, pale mucous membranes, and lethargy. Animals may be belligerent if hypoxia affects the brain. Cows in late pregnancy may abort. Animals less than 1 year old are susceptible to infection but relatively resistant to disease, whereas cattle older than 36 months of age usually experience severe disease with a 30% to 50% case fatality rate.

Anaplasmosis induced by A. marginale occurs in four stages. The incubation period comprises the time from introduction of the agent into a susceptible animal until 1% of the red blood cells are parasitized. This varies from 3 to 8 weeks and depends on the initial number of infecting organisms. During this time the animal remains asymptomatic. The first clinical signs become apparent during the developmental stage, when more than 15% of the erythrocytes are infected. Fever is the initial finding, followed by anorexia, depression, and lethargy. The length of this period ranges from 4 to 9 days. The convalescent stage varies greatly in length, from weeks to months, and extends from the appearance of reticulocytesin peripheral circulation until the blood values return to normal. Mortality may occur duringthe early convalescent stage. Necropsy findings are attributable to hemolytic anemia. Grossly, the blood appears thin and watery. Other findings include tissue pallor, icterus, hepatosplenomegaly, and enlarged gallbladder. The carrier stage may last for the remainder of the life of the animal.

Within the tick, the bacterium undergoes a complex developmental cycle that involves the gut initially and ultimately the salivary gland, from which transmission occurs during feeding. When the organism invades mature erythrocytes, replication occurs by binary fission within membrane-bound cytoplasmic inclusions and two to eight infective initial bodies are formed. These leave the red blood cells by exocytosis to infect other susceptible erythrocytes.

The severity of the disease is related directly to the proportion of the erythrocyte mass destroyed. Hemoglobinuria does not occur in anaplasmosis because the destruction of erythrocytes occurs intracellularly rather than intravascularly. Serum factors sensitize erythrocytes to phagocytosis by the monocyte-macrophage system and these opsonins increase in the circulation before the hemolytic crisis. Fetal hypoxia is responsible for abortion.

Persistence of A. marginale in fully immunocompetent hosts is mediated by antigenic variation in the organism’s major surface proteins. Recently, A. marginale has been propagated in continuous culture in a tick embryo cell line. This system has been used as an infection model and for adhesion studies. Adhesion, infection, and transmission are mediated by major surface protein 1a.

A presumptive diagnosis of anaplasmosis is based on clinical signs and hematologic findings in animals in an endemic area. Serologic testing, direct examination of blood smears, and molecular methods are also useful. Rapid card agglutination, complement fixation, and enzyme immunoassays are available for serologic diagnosis. Phenotypic criteria for identification of ruminant erythrocytic Anaplasma spp. (A. centrale, A. marginale, and A. ovis) have for many years relied on subjective methods, such as the location of inclusion bodies in host red blood cells (Figure 40-3) and host pathogenicity (cattle vs. sheep). Recentstudies have confirmed the suitability of 16S rDNA sequence analysis to define the species from blood samples.

FIGURE 40-3 Blood smear from cow with anaplasmosis, showing two typically parasitized erythrocytes and an immature form.

(Courtesy Raymond E. Reed.)