Serological Detection of Caseous Lymphadenitis in Sheep and Goats Using a Commercial ELISA in Grenada, West Indies

Abstract

Caseous lymphadenitis (CLA) in small ruminants has been diagnosed by culture several times in the state of Grenada. In order to understand the prevalence of CLA, serum samples from 541 sheep and 338 goats from Grenada and Carriacou islands were tested for antibodies against the phospholipase antigen of Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis (CLA) using a commercial ELISA kit: “ELITEST CLA”. The percentage of positive samples among sheep was 7.9, and for goats it was 31.3%, with a significant difference between these two species of animals. Of the six parishes of Grenada, and one parish of Carriacou, significantly more positive samples originated from St. George parish. There were no significant differences between age groups. The results of this study show that CLA is endemic and widely distributed throughout the islands of Grenada and Carriacou.

Keywords: Caseous lymphadenitis, sheep, goats, ELISA, Grenada

Introduction

Sheep and goats are important production animals in the state of Grenada, a tri-island nation in the Caribbean, due to their manageable size and feed requirements plus their ability to utilize sloped terrain and limited pasture areas. The rearing of sheep and goats for both meat and milk is increasing in Grenada (Annual Report, 2009). Grenada has an estimated small ruminant population of 3,000 sheep and 2,500 goats.

Caseous lymphadenitis (CLA) is a contagious and chronic disease of sheep and goats, caused by Corynebacterium pseudotuberculosis, a facultative intracellular bacterium. Abscesses, generally, involving subcutaneous lymph nodes are a common feature. Internal organs, especially lungs and internal lymph nodes can be affected. In both sheep and goats, production losses in weight gain, milk production, reproductive efficiency and carcass quality can occur (Powell, 2014). In a recent study in Canada on 152 goats, 3.9% of mortalities were attributed to CLA. Sporadic cases, including mastitis and occasional outbreaks in cattle, due to this organism have been reported (Shpigel et al., 1993). Nogradi and co-workers (2012) reported musculoskeletal C. pseudotuberculosis infection in 35 horses. C. pseudotuberculosis has zoonotic potential as well, and several cases of occupational infection, characterized by lymphadenitis, necessitating surgery, and antibiotic therapy, have been reported in humans handling infected animals, particularly, from Australia (Peel et al., 1997, Funke & Bernard, 2011). In sheep and goats, CLA has been diagnosed by cultural methods many times in Grenada. Monitoring of disease status by culturing clinical samples will not help in control of this disease because of the fact that animals may be infected without showing obvious clinical signs. Treatment is generally ineffective, although one study showed that intralesional or systemic administration of tulathromycin may be an acceptable alternative to opening, draining, and flushing of CLA lesions (Washburn et al., 2009). Vaccines can be used to reduce the number of cases and severity of the disease (Windsor, 2014). However, control and eradication can only be achieved by identification of infected animals and removal from the herd. For this purpose, serological tests, including ELISA have been used (Sutherland et al., 1987). There are no published records of this disease in the Caribbean islands or West Indies, although it has been reported to occur in USA, Canada, Argentina, and Brazil (Williamson, 2001; Arsenault et al., 2003; Estevao Belchior et al., 2007; Guimaraes et al., 2011). Recently, an ELISA based on recombinant phospholipase D (PLD), a secreted protein from C. pseudotuberculosis has been developed, and marketed under the trade name “ELITEST CLA”. As PLD is not known to be produced by any other sheep pathogenic bacteria, this ELISA is a very specific test (Hyphen Biomed com., 2014). This kit has been successfully used in Norway and Scotland for detection of CLA in small ruminants (Harkiss, 2014). The objective of this study was to generate data on prevalence of CLA in goats and sheep in Grenada by serological testing using “ELITEST CLA”, so that control measures can be implemented.

Materials and Methods

Prior to the commencement of serological testing, the records of the Veterinary Diagnostic Bacteriology Laboratory, St. George’s University, Grenada, were examined for reports of cases of CLA in sheep and goats from this country. In all cases, abscess contents were plated routinely on blood agar and MacConkey agar, and isolates suspected to be Corynebacterium pseudotuberculosis as described by Quinn et al. (1994) were confirmed by using Analytical Profile Index (API) Coryne strips. The growth on primary plating media was classified as “light”, “moderate”, and “heavy”, and “pure” or “mixed”. Three or less colonies of any morphological type were considered “insignificant”. Corynebacterium pseudotuberculosis was isolated from 11 clinical cases of small ruminants in Grenada in the past. Of the 11 consecutive culture-positive samples, 10 were from goats, and one from sheep. Six positive samples were from females, and 5 from males. Abscesses were mostly (6/11) found to occur on neck or mandibular area. Internal abscesses were found in one animal, a sheep (Table 1).

Table 1: Culture-positive Abscess content samples for Corynebacterium pseudotuberculosis from 11 small ruminants in Grenada (2004-2012)a

a Unpublished information from the Veterinary Diagnostic Laboratory, St. George’s University, Grenada
b NI: no information on location

Serum samples from 541 sheep, and 338 goats (16% of the total small ruminant population) from the islands of Grenada and Carriacou were obtained from clinically normal animals of at least 9 months of age. Animals were selected randomly from all 6 parishes of the island of Grenada, and from the island of Carriacou. The number of animals selected is in proportion to the number of small ruminants available in the particular parish/region.

“ELITEST CLA” (HYPHEN Biomed, neuville sur Oise, France) was used and interpreted according to manufacturer’s instructions for serological detection of CLA. The test detected IgG antibodies specific for the conserved C. pseudotuberculosis virulence factor, phospholipase D. The test was performed at a room temperature of 20-25°C as per the recommended assay procedure. The cut-off values were calculated as per manufacturer’s instructions.

Results

Of a total of 879 serum samples from sheep and goats tested, 149 (16.9%) were positive for CLA. Compared to sheep, significantly more goats (7.9% and 31.3% respectively) were positive for CLA (Table 2). There were significant differences in positive samples from parishes. St. George parish had the highest (43%), and St. Andrews had the lowest (4%) (Table 3). Age wise, sheep gave significantly lower positive results than goats in all age groups (Table 4). Age groups did not show significant differences in positivity.

Table 2: Sheep and goat samples positive for antibodies against CLAa

Note: ** – statistically significant at the .001 level.
a There is a statistical significant difference between the proportion of sheep and goats testing positive for antibodies against CLA (chi-square=81.01, p-value=<.001). Proportionately more goats tested positive.

Table 3: Parish/ region wise distribution of sheep and goat samples positive for antibodies against CLAa

Note: ** – indicates statistically significant at the .001 level
a Overall, sheep have significantly fewer positive than goats at each age group (9 months — 2 years: chi-square= 20.13, p-value<.001; 2-3 years: chi-square= 18.62, p-value<.001; 3-4 years: chi-square=8.89, p-value=.002; and 4-5 years: chi-square= 27.82, p-value <.001). No significant differences were seen between various age groups

Discussion

CLA is a chronic debilitating disease of global importance in sheep and goats, causing considerable economic losses due to culling of infected animals, carcass condemnation and decreased milk, wool production, reproductive efficiency, and mortality as well (Williamson, 2001; Debien et al., 2013). In the Americas, it occurs in Canada and USA (Debien et al., 2013, Powell, 2014). The prevalence of CLA in sheep in Quebec, Canada determined by presence of abscesses and positive culture was 21% (Arsenault et al., 2003). Similar estimates in Australian sheep in 2009, was 12.9% for the province of Victoria (Windsor, 2014). Recent studies in a semi-arid tropical region of India showed a prevalence of 1.3 % and 2.4 %, in sheep and goats, respectively by a specific PCR assay for C. pseudotuberculosis (Kumar et al. 2012, 2013). Serological tests for CLA have been described and developed further. Sutherland et al. (1987) described two enzyme-linked immunosorbent assays to measure antibody to the cell wall antigen and toxin antigen of C. pseudotuberculosis. Schreuder et al. (1994), used a double-antibody sandwich ELISA for eradication of CLA in two large flocks of sheep in which the disease was endemic. This ELISA was modified for use in sheep and goats, and it had a specificity of â‰¥ 98% for both species, and sensitivity of 94% for goats and 79% for sheep (Dercksen et al., 2000). Using this ELISA, 23% of small ruminants, mostly comprising of goats, in two districts of Malaysia, were found to be positive for CLA (Komala et al., 2008). Reboucas et al. (2011), described a modified ELISA for the detection of specific immunoglobulins against C. pseudotuberculosis secreted antigen. Using this assay, the seroprevalence of CLA in slaughtered sheep in the state of Minas Gerais, Brazil was estimated to be 43.7% (Guimaraes et al., 2011). The present study was possible because of the availability of a specific commercial immunoassay which was found useful in diagnosis of CLA in sheep and goats in Scotland and Norway, respectively (Hyphen biomed, 2014). Among sheep and goats in the present study, proportionately more goats tested positive for CLA, with a statistically significant difference. The reasons for the higher prevalence in goats are unknown. It is notable that 10 of 11 culture-positive samples in our diagnostic laboratory originated from goats (Personal communication, 2014). Among parishes, St. George showed the highest seroprevalence of CLA. The reasons are not understood, and needs further investigation. However, the data should be cautiously interpreted, because of the fact that number of samples collected from parishes varied. Further, farming is not organized in Grenada, and it is practically impossible to correlate differences in prevalence to animal husbandry conditions and environmental or other factors. It is hoped that the data generated in this study can be of help in designing future investigations. It is obvious that CLA is endemic in Grenada, and possibility of vaccination need to be considered in view of the fact that the herds are small, and culling all positive animals may not be practicable. The current study provides a baseline for continued screening of this disease in small ruminants in Grenada and other Caribbean islands.

Acknowledgements

The authors would like to thank V. Matthew-Belmar, E. Brathwaite, J. Coughlin, Z. Pearl, B. Read, and G. Roy for technical assistance.

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Serological Detection of Caseous Lymphadenitis in Sheep and Goats Using a Commercial ELISA in Grenada, West Indies

International Journal of Veterinary Medicine: Research & Reports

H. Hariharan¹ , K. P. Tiwari² , S. Kumthekar³ , D. Thomas⁴ , C. Hegamin-Younger⁵ , B. Edwards⁶ and R. N. Sharma⁷

Academic Editor: Bhupendra Nath Tripathi

Copyright © 2015. H. Hariharan , K. P. Tiwari, S. Kumthekar , D. Thomas, C. Hegamin-Younger, B. Edwards and R. N. Sharma. Distributed under Creative Commons CC-BY 4.0

Abstract

Serological Detection of Caseous Lymphadenitis in Sheep and Goats Using a Commercial ELISA in Grenada, West Indies

H. Hariharan1 , K. P. Tiwari2 , S. Kumthekar3 , D. Thomas4 , C. Hegamin-Younger5 , B. Edwards6 and R. N. Sharma7

Academic Editor: Bhupendra Nath Tripathi

Copyright © 2015. H. Hariharan , K. P. Tiwari, S. Kumthekar , D. Thomas, C. Hegamin-Younger, B. Edwards and R. N. Sharma. Distributed under Creative Commons CC-BY 4.0

Abstract

+Articles

+General Information

H. Hariharan¹ , K. P. Tiwari² , S. Kumthekar³ , D. Thomas⁴ , C. Hegamin-Younger⁵ , B. Edwards⁶ and R. N. Sharma⁷