Instituto Valenciano de Microbiología
(IVAMI)

Masía El Romeral
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Babesiosis (Babesia microti, B. bovis, B. divergens) - Molecuylar diagnosis (PCR); Identification of species by sequencing.

Information (11/29/14)

Babesiosis is a disease caused by protozoa of the genus Babesia intraerythrocytic transmitted by ticks. The disease affects a wide range of domestic and wild animals, which can be found many different species, and occasionally affects people, so it is considered a zoonosis. The important economic impact of babesiosis is in the cattle industry, but also may be affected pets, including horses, sheep, goats, pigs and dogs, with varying importance in different parts of the world. The main species affecting livestock are Babesia divergens, Babesia bigemina, Babesia bovis and Babesia more. In horses the main species is Babesia equi (now Theileria equi), Babesia canis in dogs, cats Babesia in felis and Babesia microti in mice. In people, most cases of babesiosis in the US are caused by Babesia microti from rodents, and in Europe by Babesia divergens and Babesia bovis from cattle. These parasites are often called piroplasms by the way, like pears (piriform), seen within the parasitized erythrocytes. Recognized species: Babesia bigemina, B. bovis, B. caballi, B. canis canis, B. canis vogeli, B. canis rossi, B. equi [Theileria equi], B. felis, B. gibsoni, B. major, B . microti, B. motasi, B. ovis, B. perroncitoi, B. trautmanni.

This disease is a zoonosis transmitted by ticks, which act as carriers hosts, and inoculate the animal reservoir during its blood meal. The main vectors are Ixodes ticks. In the US the main species involved is Ixodes scapularis (also called Ixodes dammini), while in Europe is Ixodes ricinus. The vector tick is the same as in every place transmits Borrelia burgdorferi. These ticks go through three stages in their development, larva, nymph and adult stages each loas requires a blood meal to move to the next phase. When larval and nymph feed on blood in rodents to evolve to the next stage. However, as adults, they prefer to feed on other hosts like deer in the US, or in cattle in Europe. When the tick feeds ingested infected blood, protozoan multiplies in the cells of the intestinal wall of the tick, and then concentrated in the salivary glands, where they are inoculated in the subsequent meal. When enter the bloodstream, they infected erythrocytes, becoming trophozoites, divided asynchronously resulting 2 to 4 merozoites. These depart erythrocyte membrane damage.

Human babesiosis is rare and usually asymptomatic in healthy individuals. When symptomatic generally results in a mild disease which is recovered without treatment, although it is recommended to treat to prevent damage and possible human transmission through blood transfusions. Severe disease that produces significant effect and can be fatal, occurs mostly elderly, immunocompromised and asplenic. The most common signs and symptoms are fever, and hemoglobinuria hemolytic anemia. The erythrocyte fragments can cause capillary blockage or microcapillary stasis, which explains the hepatic, splenic, renal and central nervous system involvement. Altered erythrocyte membrane increases adhesion, resulting in pulmonary edema and acute respiratory distress syndrome. Reticuloendothelial cells of the spleen removed from circulation affected erythrocytes and therefore the disease is more severe in patients.

It is a difficult disease to diagnose, and should be suspected in endemic areas with infected animals. Confirmation of the diagnosis depends on the degree of parasitemia and laboratory experience.

The laboratory diagnosis is usually made by microscopic examination of thin extensions, or thick film, stained with Giemsa o Wright. The parasitemica in individuals with spleen usually mild or moderate impairment have 10% or less of infected erythrocytes, whereas in asplenic patients can reach 85%. In erythrocytes affected forms must be observed in intraerythrocytic ring or merozoites forming tetrads, reminiscent of the Maltese cross.

Antibody class IgM or IgG may be detected by indirect immunofluorescence. An IgM titer antibodies of 1:64 or greater is considered significant. There are no cross between Babesia microti antigens and other species so the detection of anti- Banbesia microti can not be used to diagnose infections by other species reactions. Moreover, antibodies can be found in individuals who have recovered from the infection. For both reasons not recommend diagnosis through antibody detection.

Hamster inoculation (Babesia microti), or gerbil (Babesia divergens) can be performed, but require follow - up 6 to 8 week, so it is not practical for rapid diagnosis.

PCR detection is useful, correlates well with persistent antibodies, and is more sensitive than the test blood parasitemia or hamster inoculation. This test can detect very low parasitaemia, and to differentiate species by subsequent sequencing.

Tests in IVAMI:

• Molecular Detection by genomic PCR amplification followed by sequencing if desired to know the species.
• Microscopic Vision:
To be performed if required, but is less sensitive, so do not recommend it .

recommended shows

• whole blood collected with EDTA (2 to 5 mL).

Preservation and shipment of sample:

Refrigerated (preferred) for less than 2 days.
Frozen: over 2 days.

Delivery term:

• molecular targets for PCR: 48 to 72 hours.
• Identification of species by sequencing: 72 to 96 hours.

Cost of the test:

• Consult ivami@ivami.com