Abstract
Radio Immune Assay (RIA) is an extremely sensitive in vitro assay technique to measure concentrations of antigen viz. hormones in biological fluids using antibodies. The present study reports the status of total triiodothyronine (TT3) and total thyroxine (TT4) in Trypanosoma evansi infection in a dog, year 2022. An adult, non-descript, male dog was referred to the Department of Veterinary Nuclear Medicine, Mumbai Veterinary College, (MAFSU), Parel, Mumbai (India) with a history of inappetence, weakness, and ataxia of the hind limbs. Inspection revealed cachexia, anemia, bedsores, and mild mucopurulent ocular and nasal discharge. Clinical examination revealed pyrexia, polypnoea and tachycardia. There was an enlargement of popliteal, prescapular, and submandibular lymph nodes. The blood smear examination revealed severe infection of extracellular T. evansi. Laboratory investigations showed an altered haemato-biochemical profile. RIA-enabled thyroid hormone profile revealed a reduced concentration of TT3 (0.57 nmol/l) and TT4 (22.52 nmol/l). The present study reports a reduction in the concentration of TT3 and TT4 in a dog suffering from trypanosomiasis. The drop in TT4 concentration was within the normal limit, this could be a cause for the non-appearance of usual clinical symptoms of hypothyroidism in the present case.
Keywords: Canine trypanosomiasis, Triiodothyronine (TT3), Total thyroxine (TT4), Radio immune assay (RIA)
Introduction
Alterations in thyroid hormone concentrations in response to Non-Thyroid Illness (NTIs) are well recognized in veterinary clinical practice (1, 2). In severe NTIs, there is a decreased concentration of Triiodothyronine (TT3) and Total thyroxine (TT4). Trypanosoma evansi has a wide host range in most companion animals. The disease may occur in both acute and chronic forms. The reports on alterations in haemato-biochemical profile by acute and chronic infection are well documented (3, 4), However, reports on the status of thyroid function during canine trypanosomiasis have been little studied in veterinary clinical practice (5, 6).
Present clinical case record is first reported from Maharashtra (India) about the status of total triiodothyronine (TT3) and total thyroxine (TT4) in T. evansi infection in a dog.
Case Report
A Non-descript, adult, male, neutered dog was presented to the Department of Veterinary Nuclear Medicine, Mumbai Veterinary College, Parel-Mumbai 400012 (India) in the year 2022.
Institutional Biosafety committee (IBSC) approved the study based on the Declaration of Helsinki. The owner presented this dog with the complaint of non-ambulatory paraparesis. Anamnesis of the patient revealed inappetence, progressive weakness, and ataxia of the hind limbs for a fortnight. Inspection of the patient revealed evidence of cachexia, anaemia (Fig. 1 and 2), decubital ulcers/pressure ulcers (bed sores) (Fig. 3), and mild mucopurulent ocular and nasal discharge. Clinical examination of the patient revealed pyrexia (103 °F), polypnoea (29 breaths/minute), and tachycardia (102 bpm).
Fig. 1:
Pale conjunctival mucous membranes
Fig. 2:
Pale oral mucous membranes
Fig. 3:
Cachexia and bed sores
There was an enlargement of popliteal, prescapular, and submandibular lymph nodes. On neurological examination, pain sensation (deep pain) reflexes were absent in rear limbs. Abdominal palpation revealed splenomegaly and urinary incontinence. After clinical examination of the patient, blood was collected for complete blood count (CBC), liver function test (LFT), kidney function test (KFT), and thyroid profile testing (by Radio Immune Assay-RIA). Additionally, thin blood smears were also made from ear tips and stained with Giemsa stain to detect peripheral parasitemia. The results of the laboratory investigations are presented in Table 1. The microscopic examination of the blood smear revealed the presence of T. evansi (slender forms of trypanosomes with subterminal kinetoplasts, large undulating membrane, central nucleus and a long free flagellum) (Fig. 4).
Table 1:
Laboratory Investigation of a dog suffering from T. evansi infection and reference value for thyroid profile
| Sr. No. | Parameter | Result |
|---|---|---|
| 1 | Hb (gm%) | 4.30 |
| 2 | TEC (million/cmm) | 1.87 |
| 3 | PCV (%) | 12.60 |
| 4 | MCV (fl) | 67.38 |
| 5 | MCH (pg) | 22.99 |
| 6 | MCHC (g/dl) | 34.13 |
| 7 | TLC ( *103/cmm) | 3.80 |
| 8 | Platelets ( /cmm) | 22000 |
| 9 | Blood Parasites (peripheral Blood smear examination) | Trypanosoma evansi (+++) |
| 10 | BUN (mg/dl) | 20.10 |
| 11 | Serum Creatinine (mg/dl) | 1.10 |
| 12 | Bilirubin Total (mg/dl) | 0.20 |
| 13 | Bilirubin Direct (mg/dl) | 0.10 |
| 14 | Bilirubin Indirect (mg/dl) | 0.10 |
| 15 | SGPT (IU/L) | 171.00 |
| 16 | SGOT (IU/L) | 107.00 |
| 17 | Alkaline Phosphate (IU/L) | 195.00 |
| 18 | Total Proteins (gm/dl) | 6.40 |
| 19 | Albumin (gm/dl) | 2.30 |
| 20 | Globulin (gm/dl) | 4.10 |
| 21 | A/G | 0.56 |
| 22 | TT3 (nmol/l) | 0.57 |
| 23 | TT4 (nmol/l) | 22.52 |
| 24 | Reference Value TT3 (nmol/l) | 1.29 ± 0.04** |
| 25 | Reference Value TT4 (nmol/l) | 28.17±1.18** |
| 26 | TT3 (nmol/l): Interquartile range (25th to 75th percentile) | 0.88–1.51** |
| 27 | TT4 (nmol/l): Interquartile range (25th to 75th percentile) | 15.70– 35.29** |
Galdhar C.N. (2021)
Fig. 4:
Demonstrating Trypanosoma evansi
Thyroid hormones (TT3, and TT4) were estimated using RIA and the procedure was carried out at the Radio Isotope Laboratory, Department of Veterinary Nuclear Medicine, Mumbai Veterinary College, Mumbai (India). Commercial RIA kits for human purposes manufactured by the Board of Radiation and Isotope Technology (BRIT), Vashi, Mumbai (India) were procured for thyroid profile estimation. Thyroid hormones were assayed of the collected sample giving paired observation. TT3 and TT4 were measured as per the standard procedure outlined by the manufacturers. Quality control parameters viz. magnitude of control samples and recovery percentage were studied to validate the assay.
Discussion
Based on clinical and laboratory findings, the dog was suffering from T. evansi infection. In the present study, the authors are reporting the status of TT3 and TT4 in a dog suffering from T. evansi infection, for the first time from Maharashtra (India). The estimation of thyroid profile, was undertaken by Radio Immune Assay (RIA). RIA is extremely sensitive in vitro assay technique to measure concentrations of antigen viz. hormones in biological fluids using antibodies. The technique is impressively sensitive and specific with the ability to measure small molecules. I125 is the radioisotope used in RIA procedures. The method offers a convenient assay of large numbers of samples with good precision. It can assay materials that are difficult to detect and measure using other techniques (7). Since 1975, technological advances in RIA have progressively improved the specificity, reproducibility, and sensitivity of thyroid testing methods and thus RIA has been the method of choice for measuring circulating levels of thyroid hormones in vertebrates (8). Further, RIA is considered the best assay to estimate hormones as compared to other techniques viz. ELISA and CLIA (Chemiluminescence Immunoassay). RIA technique is sensitive, cost-effective and the use of long half-life radioactive materials (I125; physical half-life 60 days) facilitates its use for a long time. Though RIA is the best test to estimate hormones. However, the use of radioactive materials and their disposal is critically important. It is compulsory that assay procedures must be undertaken under the supervision of a Radiological Safety Officer (9).
In the present investigation, RIA-enabled concentrations of TT3 and TT4 were 0.57 nmol/l and 22.52 nmol/l, respectively. These estimated hormonal concentrations were lower in magnitude as compared to the reference range reported (2), Table 1. When authors apprise thyroid profile critically, a reduction in the concentration of TT3 was noticed along with a reduction in the concentration of TT4, but a drop in TT4 concentration was in the normal reference range. Therefore, no clinical signs of hypothyroidism were seen in the ailing dog. The reports on impairment in thyroidal function after experimental infection with trypanosomiasis have been documented in goats and cattle (5, 10), however, mechanisms by which trypanosomes affect thyroid functions are not completely understood.
Conclusion
The present study reports a reduction in the concentration of TT3 and TT4 in a dog suffering from trypanosomiasis. The drop in TT4 concentration was in the normal limit, this could be a cause for the non-appearance of clinical symptoms of hypothyroidism in the present case.
Acknowledgements
The authors are thankful to Associate Dean, Mumbai Veterinary College, Parel-Mumbai-India for providing the facility to undertake radiation work. Authors duly acknowledge the support of the Board of Radiation and Isotope Technology (BRIT), Department of Atomic Energy (DAE), Govt. of India for providing RIA kits.
Footnotes
Conflicts of interest
The authors declare that there is no conflict of interest.
References
- 1.Galdhar, Chandra S., Dadke A., Gaikwad R., Sarode A. Metabolic and hormonal changes in water buffaloes during post-parturient peak lactation. Buffalo Bulletin. 2021; 40 (4), 565–570. [Google Scholar]
- 2.Galdhar CN. Veterinary Applications of Radio Immune Assay (RIA) with Special Reference to Thyroid Dysfunction for Constituent Colleges of Maharashtra Animal and Fishery Sciences University (MAFSU). Project report submitted to Maharashtra Animal and Fishery Sciences University (MAFSU), Under MAFSU Research Grants (2021).
- 3.Soulsby EJL. Helminths, arthropods and protozoa of domesticated animals. 7 Delhi: Elsevier; 1982. p. 533. [Google Scholar]
- 4.Gutierrez C, Corbera JA, Juste MC, Doreste F, Morales I. An outbreak of abortions and high neonatal mortality associated with Trypanosoma evansi infection in dromedary camels in the Canary Islands. Vet Parasitol. 2005; 130(1):163–168. [DOI] [PubMed] [Google Scholar]
- 5.Abebe G, Eley RM. Trypanosome–induced hypothyroidism in cattle. Br Vet J. 1992; 148:63–70. [DOI] [PubMed] [Google Scholar]
- 6.Sudhakara Reddy, Nalini Kumari K, Sivajothi S, Rayulu VC. Haemato-biochemical and thyroxin status in Trypanosoma evansi infected dogs. J Parasit Dis. 2016; 40(2):491–495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Goldsmith SJ. Radioimmune assay: Review of basic principles. Semin Nucl Med. 1975;5: 125–152. [DOI] [PubMed] [Google Scholar]
- 8.Noyes PD, Lema SC, Roberts SC, Cooper EM, Stapleton HM. Rapid method for the measurement of circulating thyroid hormones in low volumes of teleost fish plasma by LCESI/MS/MS. Anal Bioanal Chem. 2014;406 (3): 715–726. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Galdhar CN and Gaikwad RV presented lead paper on “Radio Immune Assay (RIA): concept and application in veterinary clinical practice and research” in 35th annual convention of ISVM and National Symposium on Innovative Techniques, Emerging Issues and Advancement in Veterinary Medicine to Meet the Challenges, Present and the Future. 2017; pp. 321–324. [Google Scholar]
- 10.Mutayoba BM, Ohara-Ireri HB, Gombe S. Trypanosome–induced depression of plasma thyroxine levels in prepubertal and adult female goats. Acta Endocrinol (Copenh). 1988; 119:21–26. [DOI] [PubMed] [Google Scholar]