investigation of an outbreak of caseous lymphadenitis

Indian Journal of Small Ruminants 2018, 24(1): 95-100

Indian Journal of Small Ruminants

INVESTIGATION OF AN OUTBREAK OF CASEOUS LYMPHADENITIS IN GOATS K. Gururaj, D.D. Singh, R.V.S. Pawaiya*, Dimple Andani, N.K. Gangwar, A.K. Mishra, Vinay Chaturvedi and Ashok Kumar Division of Animal Health ICAR-Central Institute for Research on Goats, Makhdoom- 281 122, Uttar Pradesh *E-mail address: [email protected] Manuscript received on 20.01.2017, accepted on 08.06.2017 DOI: 10.5958/0973-9718.2018.00008.9

ABSTRACT Caseous lymphadenitis (CLA) is a chronic and sub-clinical disease of sheep and goats with high prevalence in domestic animals. It is caused by Corynebacterium pseudotuberculosis biovar ovis. An outbreak of CLA in superficial and visceral forms was observed in Barbari and Jakhrana goat herds maintained at ICAR-CIRG, Makhdoom (UP) in July, 2016. A total of 147 goats was affected by the infection of external lymph nodes and two cases in Barbari goats showed internal abscess on necropsy. Biopsy pus samples were collected and identified the organism after Gram's staining. All biopsy samples were inoculated to blood agar and subjected to biochemical (catalase, oxidase and nitrate) tests. Isolation of DNA from culture and 16S rRNA gene-based PCR for C. ovis was carried out for confirmatory diagnosis. Gross and histopathological studies were conducted in cases of internal abscess. Antibiotic sensitivity tests were conducted for all the isolates obtained during the study. External lymph nodes were predominantly affected involving pre-scapular and post-scapular (94), pre-femoral (44) and mandibular lymph nodes (9). After incising abscesses, white thick pus oozed out. In Grams' stained smears, the organism was pleomorphic rods arranged in Chinese letters. On blood agar, round, raised, greyish colonies were observed and biochemical examination showed catalase positive and oxidase negative. The 16S rRNA genebased PCR confirmed the presence of C. ovis. In two cases, visceral abscess was observed with oozing pus from affected lungs. Histopathology of affected lung tissue showed infiltration of inflammatory cells including neutrophils, lymphocytes and macrophages within infected tissue and concentric fibrous layers separated by inspissated caseous exudate. Based on morphology, biochemical tests and PCR, causal organism was confirmed as C. ovis and based on 16 antibiotics tested for sensitivity, ciprofloxacin was the only antibiotic found to be effective against isolates. Key words: Antibiotic sensitivity test, Caseous lymphadenitis, Corynebacterium ovis, Goat, Polymerase chain reaction

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aseous lymphadenitis (CLA) is a chronic and sub-clinical disease of sheep and goats with worldwide distribution as well as high prevalence in domestic animals. In literature, chronic suppurative lymphadenitis of external and internal lymph nodes has been described as caseous lymphadenitis and abscess disease in sheep and goats (Moussa and

Shibl, 2009; Al-Harbi, 2011). Caseous lymphadenitis caused by Corynebacterium pseudotuberculosis biovar ovis also called as C. ovis, mainly affects sheep and goats, but may infect cattle and horses, and rarely, humans; therefore, the disease has been considered an occupational zoonotic disease (Williamson, 2001). C. pseudotuberculosis is classified into two biovars

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(Biberstein et al., 1971), the biovar 'ovis', which mainly affects sheep and goats, causing superficial and visceral abscesses, and the biovar 'equi', which mainly affects horses (Connor et al., 2000, 2007). The organism has also been isolated from other species like pigs, buffaloes, deer, porcupines, llamas, camels and laboratory animals (Williamson, 2001; Dorella et al., 2006). The disease is characterized by abscess formation of superficial and visceral lymph nodes. In the superficial form, the peripheral lymph nodes swell and produce abscess, while in the visceral form there are systemic complications that can lead to chronic debility in affected animals (Radostits et al., 2007). C. ovis is easily disseminated throughout the herd by routine management practices like shearing, dipping, vaccination, etc., through environmental contamination by soil and fomites (Brown and Olander, 1987). Caseous lymphadenitis has significant economic concern for goat husbandry due to reduced meat and milk yield, loss of fertility, culling of affected animals, condemnation and downgrading of affected carcass at the time of slaughter and meat inspection (Guimaraes et al., 2011). In India, there are only a few reports on the occurrences of CLA outbreaks among the goats in Kerala (Mohan et al., 2008) and Rajasthan and India (Kumar et al., 2012; Tripathi et al., 2016). The objective of the present study was to investigate the etiology of CLA and its confirmation by PCR assay amplifying 16S rRNA genes for rapid and specific detection of C. ovis in clinical samples for proper cure in affected goats. MATERIALS AND METHODS An outbreak of CLA (superficial and visceral forms) was observed in July, 2016 in Barbari and Jakharana goats at ICAR-Central Institute for Research on Goats, Makhdoom, Uttar Pradesh. The animals affected were maintained under semiintensive system in semi-arid tropical region. A total of 818 Barbari and 214 Jakhrana goats at the farm were clinically examined for the presence of abscess of superficial lymph nodes. The females of Barbari and Jakhrana were found to be affected with abscess; however, adult males maintained in separate sheds

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were found to be healthy. The consistency of enlarged lymph nodes at various locations varied from very hard to soft and did not evince pain on palpation. The location of the affected lymph nodes was also recorded. A total 147 goats (86 Barbari and 61 Jakharana) was affected with the infection of external lymph nodes. Two cases of internal abscess were observed during post-mortem examination in Barbari goats. Biopsy samples were collected from all the abscesses for microbiological, biochemical and molecular investigation. The pus samples were aspirated aseptically from abscess lymph nodes of 147 cases using sterile disposable syringe with 18 gauge needle. The samples were carried on ice to the laboratory for further analysis. Smears were prepared from the pus samples and Gram's staining was done directly to identify any bacterial involvement. All the biopsy pus samples collected were directly inoculated on blood agar base (Himedia, Mumbai, India) supplemented with 5.0% defibrinated sheep blood and incubated aerobically at 37°C for 72 h. Isolation of DNA was done from blood agar culture plates using commercially ® available kits (QIAamp DNA Mini Kit, Germany) following manufacturer's instructions. The eluted product containing purified genomic DNA was transferred into a new 1.5 ml micro centrifuge tube and the quantity and quality of DNA was assessed at A260 nm using bio-photometer plus (Eppendorf, USA) and ° stored at -20 C until further use. A 16S rRNA genebased PCR using published primers F-5′A C C G C A C T T TA G T G T G T G T G - 3 ′ a n d R - 5 ′ TCTCTACGCCGATCTTGTAT-3′ (Çetinkaya et al., 2002) for specific detection of C. ovis was carried out for confirmatory diagnosis using an Emerald GTamp master mix (TaKaRa, Japan). The reaction conditions include 98°C for 2 min of initial denaturation followed by 30 cycles of denaturation at 98°C for 20 sec, annealing at 56°C for 20 sec and extension at 72°C for 20 sec and finished with a final extension at 72°C for 3 min. The previous isolate of C. ovis present in our laboratory repository was used as positive control.


Investigation on caseous lymphadenitis in goats

Gross and histopathology were conducted for two goats affected with internal abscess. The affected lung tissues were collected in 10% neutral buffered formalin for histopathology. Neutral buffered formalin fixed lung tissues were processed and sections were cut at 6 µ thickness and stained with hematoxylin and eosin stain following conventional procedure (Luna, 1972). After staining, the slides were observed under microscope (100X) for histological alterations. The representative isolates of C. ovis from cases of CLAwere tested for their antibiotic sensitivity as per Bauer et al. (1966). RESULTS AND DISCUSSION The overall incidence of C. ovis was 14.44% (149/1032) while it was 13.06% in Barbari and 17.04%

in Jakhrana goats (Table 1). In the present study only females were affected as males were kept separately in different sheds The incidence of C. ovis in goats was higher than the recent findings by Kumar et al. (2012) 2.4% on specific PCR assay for C. pseudotuberculosis and by Tripathi et al. (2016) - 4.38% in goats maintained at ICAR-Central Sheep and Wool Research Institute, Avikanagar (Rajasthan). In goats, the superficial lymph nodes are most affected lymph nodes (Mira et al., 2014). However, quite higher incidence was (31.30 to 48.48%) reported by Hariharan et al. (2015) in small ruminants of West Indies. The affected lymph nodes were 63.09% pre-scapular and post scapular (Plate 1), 29.53% pre-femoral, 6.04% mandibular and 1.34% had internal abscess.

Table 1. Incidence, case fatality and recovery rate for caseous lymphadenitis in Barbari and Jakhrana goats Breed

Incidence (No. affected / No. at risk)

% Case

% Case

Female

Male

Total

fatality

recovery

Barbari

16.33 (88/539)

0.00 (0/135)

13.06 (88/674)

2.27 (2/88)

97.73 (86/88)

Jakhrana

21.86 (61/279)

0.00 (0/79)

17.04 (61/358)

0.00 (0/61)

100.00 (61/61)

Total

18.22 (149/818)

0.00 (0/214)

14.44 (149/1032)

1.34 (2/149)

98.66 (147/149)

Previous reports showed that the abscess formation occurred superficially in external lymph nodes, including sub-mandibular, parotid, pre-scapular, sub-iliac, popliteal and supra-mammary lymph nodes due to caseous lymphadenitis infection (Williamson, 2001). Further, difference in the site of the abscess was also reported among sheep and goats, with the superficial form being more common in goats (Brown and Olander, 1987). The abscess was covered by thick fibrous layer and pus oozed out on incision. The consistency of pus was thick, caseous and greenish-tinged, which was similar to earlier reports of Brown and Olander (1987). Gram's staining showed pleiomorphic Gram-positive bacteria amidst mononuclear cells with the previous studies describing similar findings (Jones and Collins, 1986). In Grams' stained smears, the organism was pleomorphic rods arranged in the form of Chinese letters or typical palisade arrangement (Plate 2) which corroborated the findings of Quinn et al. (2005).

Plate1.Abscess formation and pointing in post-scapular lymph node in goat affected by caseous lymphadenitis

Plate 2. Gram's smear of pure culture of C. ovis showing filamentous rods, grouped in parallel cells as Chinese letters or 'palisade' arrangement (100X)


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It is most likely that clinical diagnosis by morphological characteristics may lead to an overestimate of incidence of CLA. It is, therefore, suggested that the suspected CLA cases must be confirmed by the bacterial culture or the molecular test for correct diagnosis. All the biopsy samples collected were inoculated to blood agar and incubated overnight at 37°C. In 142 cultured samples, bacterial colonies were round, raised, grayish with no haemolysis. Some colonies were pinpointed with a zone of alpha haemolysis (Plate 3). The occurrence of round, raised, cream-coloured colonies without haemolysis in blood agar are in agreement with the finding of Jones and Collins (1986). The non-haemolytic bacteria obtained in most cases were stained by Gram's technique which again showed pleomorphic Gram-positive bacteria arranged in palisade form like that of C. ovis. Further biochemical examination was done which showed catalase positive and oxidase negative. The organism was non-nitrate reducing in nature. These observations are supported by previous studies, wherein the estimates of CLA incidence on clinical basis have always been higher than on the bacteriological and molecular bases (Al-Harbi, 2011; Tripathi et al., 2016). For confirmatory diagnosis, 16s rRNA genebased PCR-specific for detection of C. ovis was carried out. Out of the 149 samples tested, 137 cases were confirmed as positive by PCR-based on the specific product size of 851bp (Plate 4). PCR is used to identify C. ovis, with the advantage of being faster and more specific (Çetinkaya et al., 2002). Multiplex PCR-based on amplification of the genes 16s rDNA, rpoB and pld, showing 94.6% diagnostic sensitivity, for C. ovis isolates as well as for clinical material were observed by Pacheco et al. (2007). The representative isolates of C. ovis from cases of CLA were tested for their antibiotic sensitivity (ABST) on the 16 antibiotics tested including amoxycillin, kanamycin, oxytetracycline, ciprofloxacin, ceftrioxone, ceftiofur, sulphadiazine, chloramphenicol, gatifloxacin, sparfloxacin,

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Plate 3. Growth of Corynebacterium ovis on blood agar showing round, raised, greyish-white colonies without haemolytic zone 1

2

3

4

5

bp 3000 2000 1500 1000 500

Plate 4. 16s rRNA gene-based PCR for specific detection C. ovis showing 851bp amplicon in positive samples. Wells, 1- DNA ladder, 2- Positive control (from lab. repository), 3-no template control, 4- positive unknown sample, 5- negative unknown sample

cloxacillin, gentamycin, azithromycin, cefotaxime and cefuroxime. Out of 16, only one antibiotic (ciprofloxacin) was found to be sensitive against all the C. ovis isolates tested. Based on ABST results, a course of ciprofloxacin (40 mg/ml) was administered intra-lesionally (0.5 ml single dose) and parenterally (I/M; 2.0 ml bid for 3 days) to the affected goats and all the goats having superficial CLA were cured and reduced the recurrence of new cases in the affected herd.


Investigation on caseous lymphadenitis in goats

On necropsy, the most common lesions were in the subcutaneous lymph nodes and extended to the lungs, where caseous multiple abscesses filled with yellow, thick creamy pus (Plate 5). Microscopic examination revealed a granulomatous lesion with caseous necrosis in centre of the lesion. The granulomatous lesion in the lung tissue of infected goats consisted of a collection of inflammatory cells including neutrophils, lymphocytes and macrophages within the infected tissue. The accumulation of living and dead neutrophils, macrophages, lymphocytes, multinucleated giant cells, bacteria and tissue cells around caseous necrosis in the centre comprises a granuloma with concentric layers separated by inspissated caseous liquefied material (Plate 6).

As per Williamson (2001), visceral form of C. ovis infection is characterized by abscess formation of internal organs, such as lungs, liver, kidneys, uterus, spleen and internal lymph nodes (mediastinal and bronchial) along with emaciation and weakness. The abscesses in goats have creamy, pasty caseous exudate and thin layers of fibrous tissue (Brown and Olander, 1987). Examination of histopathological sections of affected tissues with CLA showed multiple focal areas of caseous materials followed by huge number of neutrophils, macrophages, lymphocytes and surrounded by proliferating connective tissue (Mira et al., 2014). In conclusion, the outbreak of caseous lymphadenitis was diagnosed by employing laboratory tests, including isolation, identification and molecular detection. Based on this diagnosis, proper clinical management and therapeutic intervention helped in effective treatment and prevention of recurrence of the disease. ACKNOWLEDGEMENT The authors are thankful to the Director, ICAR-Central Institute for Research on Goats, Makhdoom, Uttar Pradesh, for providing necessary research facilities to conduct the study.

REFERENCES Plate 5. Gross picture of cut lung parenchyma showing thick, creamy pus oozing out from abscess

Plate 6. Microscopic section of affected lung showing lesion of abscess with concentric fibrous layers separated by inspissated caseous exudate (H&E 40X)

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