Brugia malayi

The secretome of a parasite in its definitive host can be considered to be its genome in trans, to the extent that secreted products encoded by the parasite fulfill their function in the host milieu. The ‘extended phenotype’ of the filarial parasite, Brugia malayi, is of particular interest because of the evidence that infection results in potent down-modulation of the host immune response. We collected B. malayi ‘excretory-secretory’ (BES) proteins from adult parasites and using a combination of shotgun LC-MS/MS and 2D gel electrophoresis, identified 80 B. malayi and two host proteins in BES, of which 31 (38%) were detectable in whole worm extract (BmA). Products which were enriched in BES relative to BmA included phosphatidylethanolamine-binding protein (PEB), leucyl aminopeptidase (LAP, homologue of ES-62 from the related filaria Acanthocheilonema viteae), N-acetylglucosaminyltransferase (GlcNAcT) and galectin-1, in addition to the previously described major surface glycoprotein, glutathione peroxidase (gp29, GPX-1) and the cytokine homologue macrophage migration inhibitory factor (MIF-1). One of the most abundant released proteins was triose phosphate isomerase (TPI), yet many other glycolytic enzymes (such as aldolase and GAPDH) were found only in the somatic extract. Among the more prominent novel products identified in BES were a set of 11 small transthyretin-like proteins, and three glutamine-rich-repeat mucin-like proteins. Notably, no evidence was found of any secreted protein corresponding to the genome of the Wolbachia endosymbiont present in B. malayi. Western blotting with anti-phosphorylcholine (PC) monoclonal antibody identified that GlcNAcT, and not the ES-62 homologue, is the major PC-bearing protein in BES, while probing with human filariasis sera showed preferential reactivity to galectin-1 and to processed forms of myotactin. Overall, this analysis demonstrates selective release of a suite of newly identified proteins not previously suspected to be involved at the host-parasite interface, and provides important new perspectives on the biology of the filarial parasite.

Brugia malayi lifecycle

Development and replication of B. malayi occurs in two discrete phases: in the mosquito vector and in the human. Both stages are essential to the life cycle of the parasite.

� Mosquito: The mosquito serves as a biological vector and intermediate host – it is required for the developmental cycle and transmission of B. malayi.

4. The mosquito takes a human blood meal and ingests microfilariae (worm-like sheathed eggs) that circulate in the human blood stream.

5-7 In the mosquito, the microfilariae shed sheaths, penetrate the midgut, and migrate to the thoracic muscles were the microfilariae increase in size, molt, and develop into infective larvae (L1 and L3) over a span of 7-21 days. No multiplication or sexual reproduction of microfilariae occurs in the mosquito.

8-1 The infective larvae (L3) migrate to the salivary glands, enter the proboscis and escape onto human skin when the mosquito takes another blood meal. [8]

� Human: B. malayi undergoes further development in the human as well as sexual reproduction and egg production.

1-2 The infective larvae (L3) actively penetrate the skin through the bite hole and develop into adults in the lymphatic system over a span of 6 months. Adult worms can survive in the lymphatic system for 5-15 years [9].

3. The male and female adult worms mate and the females produce an average of 10,000 sheathed eggs (microfilaria) daily [9]. The microfilariae enter the blood stream and exhibit the classic nocturnal periodicity and subperiodicity.

4. Another mosquito takes a blood meal and ingests the microfilariae. Infection depends on the mosquito taking a blood meal during a periodic episode – when microfilariae are present in the bloodstream [1] [8].

Adult worms resemble the classic nematode roundworm. Long and threadlike, B. malayi and other nematode possess only longitudinal muscles and move in an S-shape motion [ 10 ]. Adults are typically smaller than adult W. bancrofti, though few adults have been isolated. Female adult worms (50 mm) are larger than male worms (25 mm) [11].

Sections of adult Brugia sp. From a lymph node, stained with hematoxylin and eosin. (L: 200x, R: 400x)

Source: Parasite Image Library, Centers for Disease Control http://www.dpd.cdc.gov/DPDx/HTML/ImageLibrary/Filariasis_il.htm

B. malayi microfilariae are 200-275 um in length and have a round anterior end and a pointed posterior end. The microfilariae are sheathed, which stains heavily with Giemsa. The sheath is actually the egg shell, a thin layer that surrounds the egg shell as the microfilariae circulates in the bloodstream. The microfilariae retain the sheath until it is digested in the mosquito midgut [1].

B. malayi microfilariae resemble W. bancrofti and Loa loa microfilariae with minor differences that can aid in laboratory diagnosis. B. malayi microfilariae can be distinguished by the noncontinuous row of nuclei found in the tip of the tail. There are two terminal nuclei that are distinctly separated from the other nuclei in the tail, whereas the tail of W. bancrofti contains no nuclei and Loa loa microfilariae nuclei form a continuous row in the tail. B. malayi microfilariae also have a characteristic cephalic space ratio of 2:1 [1] [12]

Posterior end of B. malayi microfilariae – note the two distinctive terminal nuclei

B. malayi is one of the causative agents of lymphatic filariasis, a condition marked by infection and swelling of the lymphatic system. The disease is primarily caused by the presence of worms in the lymphatic vessels and the resulting host response. Signs of infection are typically consistent with those seen in bancroftian filariasis – fever, lymphadenitis, lymphangitis, lymphedema, and secondary bacterial infection — with a few exceptions.

Lymphadenitis, the swelling of the lymph nodes, is a commonly recognized symptom of many diseases. An early manifestation of filariasis, lymphadenitis more frequently occurs in the inguinal area during B. malayi infection and can occur before the worms mature [1].

Lymphangitis is the inflammation of the lymphatic vessels in response to infection. It occurs early in the course of infection in response to worm development, molting, death, or bacterial and fungal infection. The affected lymphatic vessel becomes distended and tender, and the overlying skin becomes erythemous and hot. Abscess formation and ulceration of the affected lymph node occasionally occurs during B. malayi infection, more readily than in Bancroftian filariasis. Remnants of adult worms can sometimes be found in the ulcer drainage [1].

Lymphangitis (left) and B. malayi filarial ulcer on medial portion of the thigh

The most obvious sign of infection, elephantiasis, is the enlargement of the limbs. A late complication of infection, elephantiasis is a form of lymphedema and is caused by repeated inflammation of the lymphatic vessels. Repeated inflammatory reactions causes vessel dilation and thickening of the affected lymphatic vessels, which can compromise function. The lymphatic system normally functions to maintain fluid balance between tissues and the blood and serves as an integral part of the immune system. Blockage of these vessels due to inflammatory induced fibrosis, dead worms, or granulomatous reactions can interfere with normal fluid balance, thus leading to swelling in the extremities [13]. Elephantiasis resulting from B. malayi infection typically affects the lower extremities of the legs and arms. Unlike bancroftian filariasis, B. malayi rarely affects genitalia and does not cause funiculitis, orchitis, epididymitis, hydrocele, or chyuria, conditions more readily observed with bancroftian infection [1].

Elephantiasis in the right arm and right leg of a patient from a remote island in the sea of Korea. Patient had suffered from recurrent swelling for more than 30 years before the onset of elephantiasis.

Source: Tai Soon Yong, Web Atlas of Medical Parasitology, http://atlas.or.kr/atlas/alphabet_view.php?my_codeName=Brugia%20malayi

Secondary bacterial infection

Secondary bacterial infection is common among patients with filariasis. Compromised immune function due to lymphatic damage in addition to lymph node ulcerations and abscesses exposure and impaired circulation due to elephantiasis can cause secondary bacterial or fungal infection. Elephantiasis, in addition to the physical burden of a swollen limb, can be a severely dehabilitating condition given bacterial infection. Part of the WHO�s Strategy to Eliminate Lymphatic Filariasis targets hygiene promotion programs in order to alleviate the suffering of affected individuals (see Prevention Strategies) [1] [14].

However, clinical manifestations of infection are variable and depend on several factors, including host immune system, infectious dose, and parasite strain differences. Most infections appear asymptomatic, yet vary from individual to individual. Individuals living in endemic areas with microfilaremia may never present with overt symptoms, whereas in other cases, only a few worms can exacerbate a severe inflammatory response [1].

The development of the disease in humans, however, is not well understood. Adults typically develop worse symptoms, given the long exposure time required for infection. Infection may occur during childhood, but the disease appears to take many years to manifest. The incubation period for infection ranges from 1 month to 2 years and typically microfilariae appear before overt symptoms. Lymphedema can develop within six months and development of elephantiasis has been reported within a year of infection among refugees, who are more immunologically naive. Men tend to develop worse symptoms than women [14].

Diagnostics

Tender or enlarged inguinal lymph nodes or swelling in the extremities can alert physicians or public health officials to infection. With appropriate laboratory equipment, microscopic examination of differential morphological features of microfilariae in stained blood films can aid diagnosis – in particular the examination of the tail portion, the presence of a sheath, and the size of the cephalic space [1]. Giemsa staining will uniquely stain B. malayi sheath pink [12]. However, blood films can prove difficult given the nocturnal periodicity of some forms of B. malayi.

PCR based assays are highly sensitive and can be used to monitor infections both in the human and the mosquito vector. However, PCR assays are time-consuming, labor intensive and require laboratory equipment. Lymphatic filariasis mainly affects the poor, who live in areas without such resources [15].

The ICT antigen card test is widely used in the diagnosis of W. bancrofti, but commercial antigens of B. malayi have not been historically widely available. However, new research developments have identified a recombinant antigen (BmR1) that is both specific and sensitive in the detection of IgG4 antibodies against B. malayi and B. timori in ELISA and immunochromatographic rapid dipstick (Brugia Rapid) test. However, it appears that immunoreactivity to this antigen is variable in individuals infected with other filarial nematodes [16]. This research has led to the development of two new rapid immunochromatographic IgG4 cassette tests – WB rapid and panLF rapid – which detect bancroftian filariasis and all three species of lymphatic filariasis, respectively, with high sensitivity and selectivity [15].

Management and Therapy

The Global Program to Eliminate Lymphatic Filariasis was launched by the World Health Organization in 2000 with two primary goals: 1) to interrupt transmission and 2) to alleviate the suffering of affected individuals. Mass drug treatment programs are the main strategy for interrupting parasite transmission, and morbidity management, focusing on hygiene, improves

the quality of life of infected individuals [14].

A goal of community base efforts is to eliminate microfilariae from the blood of infected individuals in order to prevent transmission to the mosquito. This is primarily accomplished through the use of drugs. The treatment for B. malayi infection is the same as for bancroftian filariasis. Diethylcarbamazine (DEC) has been used in mass treatment programs in the form of DEC-medicated salt, as an effective microfilaricidal drug in several locations, including India [17]. While DEC tends to cause adverse reactions like immediate fever and weakness, it is not known to cause any long-term adverse drug effects. DEC has been shown to kill both adult worms and microfilariae. In Malaysia, DEC dosages (6 mg/kg weekly for 6 weeks; 6 mg/kg daily for 9 days) reduced microfilariae by 80% for 18-24 months after treatment in the absence of mosquito control [1]. Microfilariae numbers slowly return many months after treatment, thus requiring multiple drug doses over time in order to achieve long-term control. However, it is not known how many years of mass drug administration is required to eliminate transmission. But currently, there have been no confirmed cases of DEC resistance [17 ] .