Taxonomy and pathogenicity of trypanosome
Trypanosomes are protozoan parasites that inhabit the blood circulation of both humans and animals, causing various diseases Classified under the subkingdom PROTOZOA and phylum SARCOMATIGOPHORA, they belong to the class ZOOMASTIGOPHOREA, order KINETOPLASTIDA, family TRYPANOSOMATIDAE, and genus TRYPANOSOMA These parasites can be categorized into two main groups: the Stercoraria, which includes subgenera such as Schizotrypanum, Megatrypanum, and Herpetosoma, where metacyclic forms are produced in the hindgut and transmitted through contaminated feces; and the Salivaria, encompassing subgenera like Duttonella, Nannomonas, and Trypanozoon, where transmission occurs via inoculation through the anterior station.
The clinical and pathological responses to trypanosome infection in domestic animals have been extensively researched, revealing that many manifestations of trypanosomosis are similar across species However, the severity and range of pathological effects are influenced by various factors, including the specific trypanosome species involved Distinct pathological changes can occur depending on the type of livestock-infective trypanosome, with tissue pathology linked to the parasites' ability to invade extravascular spaces and organs For instance, T congolense primarily remains within the vascular system, highlighting the complexity of these infections.
The Trypanozoon group comprises two trypanosome species, T b brucei and T evansi, along with T equiperdum and T vivax, which are found in both the bloodstream and tissues of hosts Notably, there is significant intra-species variation in pathogenicity among different parasite stocks, particularly those isolated from various geographical regions, with some East African isolates exhibiting distinct characteristics.
T vivax can lead to an acute hemorrhagic disease in cattle, unlike the milder non-hemorrhagic form associated with most West African isolates Various host factors influence disease severity, with African wildlife typically exhibiting greater resistance compared to domestic ruminants and often acting as reservoirs for trypanosomes that infect humans and livestock Additionally, the host's physiological condition, along with nutritional and environmental factors, significantly affects the severity of trypanosomosis.
Nagana, surra, and dourine are three recognized animal syndromes caused by pathogenic trypanosomes Nagana, primarily affecting ruminants, camels, equines, swine, and carnivores in Africa, is caused by T congolense, T vivax, T simiae, T b brucei, or T suis In South America, T vivax predominantly affects cattle, but other livestock such as sheep, goats, horses, and water buffaloes can also be infected The incubation period for nagana typically ranges from 1 to 3 weeks, influenced by the virulence of the trypanosome, the infective dose, and the host's immune status The acute phase of the disease is marked by a high level of trypanosomes in the blood (10³ - 10⁸/ml), accompanied by remittent fever and the onset of anemia Additional symptoms include enlarged lymph nodes and spleen, along with weakness, lethargy, and loss of condition, which may lead to abortion in affected animals.
Reduced milk production is a common symptom of acute trypanosomosis, which can lead to the death of infected animals within weeks or months In some instances, the clinical condition may stabilize after 6-8 weeks, initiating a slow recovery process However, many animals progress to a chronic phase characterized by stunting, wasting, and infertility This chronic phase can persist for months or even years, often concluding with the animal's death.
Surra, caused by T evansi, is a significant type of animal trypanosomosis prevalent in Asia, South America, and Africa It can manifest acutely in young animals and pregnant females, often resulting in death within weeks, while the chronic form, common in endemic regions, can persist for years and lead to cachexia and mortality Survivors of chronic infections may eventually self-cure Key clinical signs include intermittent fever, anemia, emaciation, edema, conjunctivitis, lacrymation, enlarged lymph nodes and spleen, impaired motor function, and abortions In camels, T evansi infections may also lead to pulmonary complications, whereas cattle in South America often remain asymptomatic carriers Water buffaloes can experience acute or sub-acute infections, with a mortality rate of 5-15% occurring within a few weeks.
Dourine, a venereally-transmitted disease caused by T equiperdum, is the last type of animal trypanosomosis affecting equines This disease is widely distributed across North Africa, the Middle East, Eastern Europe, South America, and Central and Southeast Asia Horses are particularly vulnerable to T equiperdum, often succumbing to the disease after a chronic course lasting 1-2 years, though acute forms can lead to death within 2-3 months In contrast, donkeys and mules may experience milder symptoms or remain asymptomatic.
Dourine in horses has an incubation period ranging from one week to six months, often presenting in three clinical phases The initial phase features edema in the genital area, which may extend to the ventral abdominal wall Approximately 30-40 days post-infection, the second phase emerges, marked by urticarial plaques on the skin and lymph node enlargement, accompanied by symptoms such as fever, anemia, weight loss, and potential abortion As the infection progresses, neurological signs intensify, leading to a paralytic syndrome, with affected animals typically exhibiting severe emaciation and anemia before death.
Disease impact
Animal trypanosomosis is a significant global concern, with its widespread distribution highlighting its substantial impact Recent research has greatly expanded our understanding of the disease's effects, particularly on cattle production in rural and pastoral areas Numerous studies have quantified its influence on agricultural development, revealing both direct and indirect impacts on livestock.
The direct effects of disease in livestock are evident through increased mortality rates, reduced calving rates, and declines in milk and meat production, alongside issues like weight loss and infertility Research by Kristjason et al (1999) estimates that the economic impact of these diseases on livestock producers and consumers in Africa reaches approximately $1.34 billion each year Additionally, studies by Swallow (2000) and Shaw (2009) indicate that calf mortality rates in infected populations are 6-10% higher compared to healthy counterparts.
Surra has a significant impact on water buffalo populations, leading to increased mortality rates in older animals by 2-8% and reduced annual calving rates by 7% Milk yields are also affected, with reductions ranging from 2-26% A study by Dargantes et al (2009) in the Philippines found that female buffaloes comprised 69% of the surveyed population In areas with low risk, female mortality during peak reproductive years was less than 1%, while it reached 10% in high-risk areas, where the annual calving rates were 15% and 47%, respectively Consequently, surra has resulted in substantial financial losses in high-risk regions.
The indirect effects of livestock diseases, such as trypanosomosis, significantly influence agricultural practices, including breed selection, herd size, and migration patterns In sub-Saharan Africa, particularly in West Africa, Ethiopia, and southern Africa, work animals are extensively utilized despite the disease's prevalence For instance, in Ethiopian villages, the proportion of work oxen in endemic areas is notably lower—around 30%—compared to nearly 60% in non-endemic regions This discrepancy is reflected in the annual replacement of adult males in unprotected villages, highlighting higher mortality rates Different cattle breeds exhibit varying susceptibility to trypanosomes; indigenous breeds tend to be trypanotolerant but are less productive than susceptible breeds, posing economic challenges for farmers An increase in cattle populations would also positively affect crop production through animal traction and manure use Swallow (1997) estimated that a 1% rise in livestock numbers could lead to a 0.2% increase in overall agricultural output, underscoring the importance of livestock health in enhancing agricultural productivity.
On the other hand, decrease of animal population due to trypanosomosis would harmful to the agriculture.
Diagnosis importance
Diagnosis is crucial for managing animal trypanosomosis, with rural confirmations often being retrospective; thus, blood samples taken during treatment are vital for monitoring infection rates Larger farms require routine diagnostic sampling to aid disease management, while district and provincial levels depend on accurate diagnosis for effective disease surveillance and control program assessment The diagnostic process is challenging due to nonpathognomonic clinical signs and low parasitemia According to OIE, confirming the disease involves detecting parasites in the blood and conducting hematological, biochemical, and serological tests Although clinical signs provide some indication, laboratory confirmation is necessary, utilizing methods for pathogen identification and serological testing.
The identification of the pathogen involves various methods, including parasitological tests, antigen detection through enzyme-linked immunosorbent assay (ELISA), and molecular techniques like polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) During the acute phase or when there is a high level of parasitemia in the animal's blood, examining wet blood films, stained blood smears, and lymph node material can effectively reveal the presence of trypanosomes.
In chronic cases, such as the carrier state, it is advisable to conduct examinations of thick blood smears, parasite concentration, and experimental animal inoculation Advanced diagnostic methods like ELISA, PCR, and LAMP have been widely evaluated and demonstrate high sensitivity and specificity PCR detection methods can identify as few as 10 parasites per milliliter of blood, while loop-mediated isothermal amplification assays can detect just 0.1 parasites per milliliter, making these techniques highly effective for diagnosing parasitic infections.
Serological tests play a crucial role in diagnosing animal trypanosomosis by detecting specific antibodies to trypanosomes Laboratory methods such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) are commonly used For field applications, the card agglutination test for trypanosomosis (CATT) and latex tests are available However, a significant challenge with these serological tests is their inability to differentiate between current and past infections, as well as potential cross-reactions among different trypanosome species The CATT, designed for diagnosing West African human sleeping sickness, relies on the presence of anti-trypanosomal antibodies to agglutinate trypanosomes, but its effectiveness is limited in hot, dry climates due to non-specific agglutination In contrast, the IFAT and ELISA have proven to be the most reliable serological methods for diagnosing trypanosomosis, with ELISA showing comparable results to IFAT.
Local treatment history reveals that testing systems, while essential for disease surveillance, necessitate costly equipment like fluorescent microscopes or microplate readers Additionally, the results from these tests typically provide only a preliminary indication of pathogen exposure Despite these limitations, they play a crucial role in monitoring animal populations before and after control measures are implemented.
4 Objectives of the present study
This study focused on creating a rapid and accurate serological diagnostic test for detecting animal trypanosomosis Initially, a novel diagnostic antigen was identified and produced The antigen's sensitivity and specificity were evaluated using ELISA, leading to the development of an immunochromatographic test The effectiveness of the newly developed test was then assessed and validated with both experimental and field-derived samples.
Chapter 1 Recombinant TeGM6-4r as potential diagnostic antigen for animal trypanosomosis
The diagnosis of animal trypanosomosis primarily depends on identifying the parasites, as noted by the OIE in 2012 However, the sensitivity of traditional parasitological methods is often hindered by low levels of parasitemia in infected animals Although molecular diagnostic assays have been created, many of these methods remain unvalidated, according to Desquesnes and Davila (2002) and Thekisoe et al.
2005) Therefore, serological diagnosis is still retained a common method
The OIE recommends using trypanosome lysate antigen in ELISA for diagnosing surra; however, the quality of this antigen is challenging to standardize due to varying laboratory protocols, leading to high cross-reactivity and increased false-positive results In contrast, recombinant antigens offer significant advantages, including greater specificity, efficiency, and no need for animal testing Notably, the recombinant variable surface glycoprotein RoTat1.2 has been successfully expressed in insect cells and shows comparable results to the native antigen in serological tests for T evansi infection in dromedary camels However, it is ineffective in detecting T evansi type B, which does not express RoTat1.2.
10 promising recombinant protein is the invariant surface glycoprotein 75 (ISG75) presenting in approximately 5 x 10 4 molecules on trypanosome cell surface (Tran et al., 2009)
Furthermore, tandem repeat (TR) proteins have been recently proposed by Goto et al., 2010 and Nguyen et al., 2012
Tandem Repeat (TR) proteins of trypanosomatid parasites are often targets of B cell responses (Goto et al., 2007; Kemp et al., 1987; Reeder and Brown, 1996) A number of
Leishmania species and T cruzi TR proteins have been shown to have significant immunological dominance, with rK39 of Leishmania and T cruzi proteins B13, CRA, TcD, and TcE effectively utilized in disease serodiagnosis Additionally, T brucei contains numerous proteins with large TR domains, and recently identified TR proteins have demonstrated reactivity to sera from trypanosome-infected mice Notably, recombinant TbbGM6 from T brucei has shown high antigenicity in water buffalo infected with T evansi, highlighting its role as a cytoskeletal protein critical during the early infection phase Due to its conservation among trypanosomes, GM6 presents a promising diagnostic marker for general animal trypanosomosis To enhance the antigenicity of the recombinant protein, GM6 was produced with four TR domains, as opposed to the previous two.
In a study conducted in 2012, GM6 was derived from the genomic data of T evansi and was designated as TeGM6-4r, consisting of four repeats The antigenicity of the newly expressed TeGM6-4r was assessed using 15 serum samples collected from three water buffaloes experimentally infected with T evansi.
The TeGM6-4r gene was successfully cloned and sequenced by amplifying it through conventional PCR, utilizing genomic DNA from the T evansi Tansui strain The amplification employed a specific primer set, with the forward primer 5’-GGA TCC ATG GAG CTT GCT AAA-3’ and the reverse primer 5’-GAA TTC CTA ATG TGA ATG CTC-3’, which include Bam HI and Eco RI restriction sites The PCR reaction mixture consisted of 50 μl, incorporating 1.5 mM MgCl2, 2 mM of each dNTP, and 5 pmol of each primer.
The study utilized 1 unit of Taq DNA polymerase (Invitrogen Japan, Tokyo) to conduct 30 PCR cycles, involving denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 1.5 minutes The resulting PCR products, which contained varying numbers of repeat units, were separated by agarose gel electrophoresis DNA fragments with 4-repeat domains were extracted from the gel using Japan-QIAGEN K.K protocols and ligated into the pCR2.1 vector before transforming into E coli DH5α through the TA cloning method Following digestion with Eco RI restriction enzyme, the inserts underwent direct sequencing with PCR primers, BigDye Terminator Ready Mix (Applied Biosystems, Life Technologies, Carlsbad, CA), and an ABI Prism 3100 Genetic Analyzer The nucleotide and amino acid sequences were subsequently identified and analyzed using Genetyx version 8.0 (Genetyx Co., Tokyo).
12 and BLAST (http://blast.ncbi.nlm.nih.gov/)
The recombinant tandem repeat protein TeGM6-4r was expressed following the method outlined by Goto et al (2006) In summary, the gene fragment for TeGM6-4r was cloned into the pET-28a vector from EMD Biosciences, and subsequently transformed into E coli BL21 for protein expression.
BL 21 was cultured in SOB medium to an OD600 of 0.4-0.6, and the expression of the recombinant protein was initiated by adding 1 mM IPTG for 3 hours The recombinant TeGM6-4r was purified in soluble form using Ni-NTA agarose according to the manufacturer’s instructions The protein's integrity and purity were assessed using 12% SDS-PAGE, while the concentration was measured using a BCA assay The purified recombinant protein was stored at -80°C until further use.
The enzyme-linked immunosorbent assay (ELISA) was utilized to screen recombinant TR antigens, following the procedures outlined in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE, 2008) Each well of a Maxisorp microplate (Thermo Fisher Scientific, Nalgene-Nunc, Rochester, NY) was coated with 200 ng of the TR antigen and incubated at room temperature for 4 hours After incubation, the antigen-coated wells were washed five times with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS-T), followed by a wash with PBS and incubation with a blocking solution of PBS-T containing 1% bovine serum albumin.
13 serum albumin (BSA)) for overnight at 4 o C Serum samples diluted at 200 times with PBS-