Colloidal Gold-based Immunochromatographic Assay For The Diagnosis Of Encephalitozoon Cuniculi Infection Of Farmed Foxes

Microsporidia belongs to the phylum Microspora,a group of obligate intracellular parasites.This parasite has been found to infect a wide host distribution,resulting in a number of diseases(e.g.,gastroenteritis,encephalitis,and nephritis).The farmed blue fox(Alopex lagopus)is particularly susceptible to congenital infections of the microsporidian species Encephalitozoon cuniculi.Generally only fox pups display symptoms,such as stunted growth,blindness and occasionally,sudden premature death.Infection in animals is usually subclinical,but severe neurological signs can occur in foxes because of granulomatous encephalitis.Signs of disease only develop in pups.Typically,the disease affects several pups of the same litter causing heavy losses among young animals infected antepartum.The parasite localizes in the central nervous system causing ataxia,paresis of limbs,convulsions and blindness.The pups appear thirsty because of kidney lesions.Most affected foxes show reduced appetite and stunted growth,and some animals may die suddenly.An encephalitozoonosis outbreak in the blue fox with a high mortality rate and substantial economic loss has been documented.The sequence encoding SWP1 was cloned from the genome of E.cuniculi.Recombinant SWP1(rSWP1)was expressed in Escherichia coli and used to detect E.cuniculi infections in farmed foxes and dogs with an indirect enzyme-linked immunosorbent assay(ELISA).Various laboratory methods are currently available for the detection and surveillance of E.cuniculi,and include Masson’s trichrome staining,enzyme linked immunoabsorbent assay(ELISA)and PCR.However,these assays are laborious and time consuming.It is needed to develop a rapid,simple,accurate and cost-effective method which could be used for the differentiation and identification of E.cuniculi in all levels of laboratories.The splenocyte of BALB/c mice immunized with purified fox IgG were fused with SP2/0 cells.Three hybridomas secreting monoclonal antibodies(mAb)against fox IgG,designated as E2,1E3,2G10,were selected with indirect ELISA and cloned by the method of limiting dilution.Western Blot results showed that these three monoclonal antibodies can specifically react with fox IgG.Monoclonal antibodies secreted from hybridomas E2,1E3,2G10 were identified to be subclass antibodies IgG2,Kappa.Western Blot results showed that the purified ascites monoclonal antibody titers up to 6000.The sequence encoding SWP1 directionally cloned into the pColdIII expression vector.The plasmids were then transformed into E.coli BL21 competent cells to express recombinant proteins fused with GST.The rSWP1 were purified from isopropyl-D-thiogalactopyranosideinduced cell cultures using,according to the manufacturer’s instructions.Monoclonal antibody which was secreted by 1E3 hybridomas and microsporidia positive sera were used to detect the activity of SWP1 protein,ELISA results showed that SWP1 could specifically react with microsporidian antibody.The sensitivity and specificity of the ELISA based on rSWP1 suggest that this could be an alternative method for the diagnosis of E.cuniculi infections in foxes.After the preparing conditions were established,colloidal gold of 20 nm was prepared with reducing reaction using trisodium citrate.the optimal mount of monoclonal antibody which was secreted by 1E3 hybridomas was 48μg/mL.The colloidal gold labeled 1E3 monoclonal antibody diluted to 15 times was dispensed onto the conjugate pad.Rabbit anti-fox IgG polyclonal antibody was dispensed at the upper position as the control line with a concentration of 1.6 mg/mL,while the rSWP1 was dispensed on the bottom of the MILLIPORE135 membrane as the test line with a concentration of 1.0 mg/mL,so two band colors will be visible when the samples contain target antibody,otherwise,a negative sample will result in the formation of just one band color on control line.The sera of foxes infected with E.cuniculi could be distinguished from the sera of foxes infected with Toxoplasma gondii,Neospora caninum,and Cryptosporidium parvum using the test strip.Repetitive test results showed that the test strip had good reproducibility.The sera of foxes infected with E.cuniculi diluted to 100 times can be detected.After 4 months,the sealed test strip which was stored at 4?C and-20?C was valid,while the 37 ?C placed test strip was invalid.The results of the present evaluation show that the test strip is a rapid,reproducible,sensitive and simple diagnostic test for the detection of the sera of foxes infected with E.cuniculi.The sensitivity and specificity of the test strip based on monoclonal antibodiy against fox IgG suggest that this could be an alternative method for the diagnosis of E.cuniculi infections in foxes.