A primary Corynebacterium pseudotuberculosis low dose infection in alpacas (Lama pacos) protects against a lethal challenge exposure

Small Ruminant Research 72 (2007) 81–86

A primary Corynebacterium pseudotuberculosis low dose infection in alpacas (Lama pacos) protects against a lethal challenge exposure W. Braga a,b,∗ , S. Schul b , A. Nu˜nez a , D. Pezo a , E. Franco a a

Instituto Veterinario de Investigaciones Tropicales y de Altura IVITA, Facultad de Medicina Veterinaria, Universidad Nacional Mayor de San Marcos, San Borja, Lima, Per´u b Kansas State University, Diagnostic Medicine/Pathobiology, Manhattan KS 66506, USA Received 21 October 2005; accepted 4 April 2006 Available online 26 September 2006

Abstract Corynebacterium pseudotuberculosis is the agent of alpaca’s lymphadenitis. The present study was to demonstrate the effect of a primary infection with low (1.1 × 103 ), moderate (1 × 104 ), and high (1.2 × 105 ) doses of C. pseudotuberculosis against a significant higher challenge dose of 9 × 108 CFU of C. pseudotuberculosis. Three groups of 4 healthy male alpacas were inoculated subcutaneously (SC) in the left flank behind the costal arch with the above doses of bacteria. A fourth group of 4 alpacas was sham inoculated with phosphate buffered saline as control. After 5 weeks all animals were challenged with a dose of 9 × 108 CFU of C. pseudotuberculosis inoculated SC in the right flank. The alpacas were clinically inspected for local and regional abscesses, body temperature and behavior changes. The primary infected alpacas had a febrile response, and abscesses at the inoculation point and regional lymph nodes. However, after challenge, the primary infected animals showed no superficial lesions or febrile response. In contrast, the immune na¨ıve alpacas from group D developed a severe disease characterized by fever, abscesses in regional lymphnodes, and in one alpaca a subcutaneous edema and sudden death 2 weeks after exposure. In addition, primary infected alpacas had a robust antibody response against C. pseudotuberculosis cell wall antigen with significant differences with respect the na¨ıve challenged alpacas. At necropsy, the primary infected alpacas had abscesses only in the regional or internal renal-lymph nodes from the left or primary inoculation side of the body, with no lesions in the right challenged side. In contrast, the primary sham inoculated alpacas had abscesses in the regional and internal lymph nodes from the right challenged side. This work showed that a primary infection with at least 1.1 × 103 viable C. pseudotuberculosis induces protection against a second high dose exposure to this bacterium. These results will be useful for further study of prevention methods to control lymphadenitis in alpacas. © 2006 Elsevier B.V. All rights reserved. Keywords: Alpacas; Corynebacterium pseudotuberculosis; Caseous lymphadenitis

1. Introduction

∗

Corresponding author at: 1800 Denison Avenue (Coles Hall 309), Manhattan, KS 66506, USA. Tel.: +1 785 532 4620; fax: +1 785 532 48 51. E-mail address: [email protected] (W. Braga). 0921-4488/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2006.04.017

Corynebacterium pseudotuberculosis is associated with a severe granulomatous lymphadenitis in South American camelids. This chronic infectious disease is characterized as a progressive enlargement of the sublumbar or renal lymph nodes (Braga, 1993). In alpacas under natural conditions of extensive management in

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the Andes, there is no opportunity to treat each abscess case, especially if it is localized internally. In intensive production of alpacas in USA, most of the abscesses in young alpacas are localized in the head, cervical or thoracic area (Anderson et al., 2004). This localization of abscesses was described also for young alpacas under extensive management in the Andes (Villena, 1985).In sheep and goats the disease produced by C. pseudotuberculosis is known as caseous lymphadenitis (CLA), and is characterized by pyogranulomas in superficial and visceral lymph nodes, and organs, especially the lungs (Batey, 1986). Susceptibility to CLA has been demonstrated by experimental infection in sheep; by the inoculation of the external ear (P´epin et al., 1988), intralymphatic (Burrell, 1978), or intradermal inoculation (Brogden, 1984). In goats, the inoculation by the intradermal and intravenous route resulted in mediastinal and lung abscesses (Brown et al., 1985). Control of this disease in lambs and goats was achieved with vaccines containing different antigen or viable bacteria compositions. The filtrate vaccine immunization resulted in antibody responses to numerous cellular antigens in lambs (Ellis et al., 1991). Inactivated whole-cell antigen and detoxified exotoxin of C. pseudotuberculosis has been developed and evaluated for vaccine potential, and reduced the prevalence of internal abscesses (Tachedjian et al., 1995; Hodgson et al., 1999). The potency of a toxoid vaccine for the use in alpacas was assayed in mice with 80% of protection after a challenge IV lethal dose of C. pseudotuberculosis from alpaca origin (Braga et al., unpublished data). In goats, small doses of live C. pseudotuberculosis prevented the development of abscesses in the draining area or in internal organs (Johnson et al., 1993). The purpose of this study was to evaluate whether or not a primary C. pseudotuberculosis low infection in alpacas would be protective against a 10× challenge exposure, and if this protection is dose-dependant. This study will serve to elucidate this disease in alpacas in order to further develop a preventive method of control. 2. Materials and methods 2.1. Animals Sixteen, 2 year-old male alpacas destined for slaughter, originating from healthy flocks in which CLA had never been observed were used. The animals were raised together for 6 weeks before the experimental infection,. The alpacas were then weighed and housed at random in 4 pens of 6 m × 20 m, with four individuals each. They were provided with hay/ryegrass and water ad libitum. To sacrifice the alpacas by exanguination, they were anesthetized first with a combi-

nation of Xylazine–Ketamine at 0.5 mg/kg and 5 mg/kg IM, respectively (Riebold et al., 1989; DuBois et al., 2004). 2.2. Experimental primary infection and challenge A 10 passaged strain of C. pseudotuberculosis H0676 of alpaca origin was used (Braga, 1993). Organism preserved in Kliger agar at 4 ◦ C (Difco Laboratories) were reconstituted and grown for 48 h in brain–heart infusion agar (BHI agar—Diagnostics Pasteur) supplemented with 5% ovine desfibrinated blood, and 0.05% Tween 80 (Sigma Laboratories Inc.). The colonies were suspended in phosphate buffered saline pH 7.2 (PBS) and groups A, B and C were inoculated SC in the left flank, behind the costal arch with 0.5 ml/dose suspensions of 1.1 × 103 (low dose), 1 × 104 (moderate dose), and 1.2 × 105 (high dose) CFU of C. pseudotuberculosis, respectively. A fourth group D was sham inoculated with PBS as a control. Five weeks later all alpacas were challenged with a dose of 9 × 108 × 0.5 ml of C. pseudotuberculosis inoculated SC in the right flank, behind the costal arch and observed 5 weeks more. Bacterial density was estimated by the McFarlane method and plate counts. After challenge, bacteria counts revealed that the 0.5 ml inoculums contained 9 × 108 CFU. 2.3. Clinical observations and sample collection Fifteen days before the inoculation the alpacas were weighed and blood and serum were collected. After the primary infection, alpacas were observed for 5 weeks. Rectal temperatures were recorded daily for the first week. Blood samples for hematologic examination were drawn once a day from the first to the fourth day post-inoculation (PI), then once a week for 1 month. For bacterial culture examination, swabs were taken from inoculation sites which suppurated. Serum for ELISA was collected at weeks 0, 1, 2, 3 and 4 post primary infections and at weeks 5, 6, 7, 8, 9 and 10 after challenge exposure. Swabs for microbiological analysis were preserved in Cary & Blair transport medium (Difco laboratories, BD Co., Maryland, USA) no longer than 20 h and then inoculated onto BHI blood agar. All C. pseudotuberculosis isolates were identified using the API System (API Coryne, Merial, France). 2.4. Pathology All alpacas were sacrificed at week 10 post-challenge, except one alpaca each from groups A, B and C, which were sacrificed at week 9. The carcasses from alpacas that died or were euthanized from group D (control) were inspected at necropsy. Lesions observed at necropsy were sampled for bacterial examination. 2.5. Preparation of antigen for ELISA analysis The cell wall of C. pseudotuberculosis H0676 was used as the antigen, following the protocol described, with some modifications (Laak et al., 1992). Briefly, two colonies grown for

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48 h at 37 ◦ C on BHI blood agar were emulsified in 1 l of BHI broth containing 0.1% Tween 80. The broth was incubated 72 h at 37 ◦ C on a magnetic stirrer and stored overnight at 4 ◦ C. The culture was transferred to Teflon tubes (Nalgene, Rochester NY, USA) and centrifuged at 4000 × g for 15 min at 4 ◦ C. Each pellet was re-suspended in PBS pH 7.4 and centrifuged at 12,000 × g for 15 min at 4 ◦ C, 3 times (Eppendorf 5402, Hamburg, Germany). Thereafter, pellets were re-suspended in aliquots of 1.5 ml PBS pH 7.4 and stored at 4 ◦ C for 24 h. The cells were sonically disrupted (Fisher sonic cell disrupter, Pittsburg, PA, USA) at 60% capacity, 2 times for 10 min each at 4 ◦ C, extracted with diethyl ether for 3 h, and sonically disrupted again for 10 min total at 4 ◦ C. Final protein concentration was determined by the Bradford method (Bio-Rad protein assay kit II, Hercules, CA, USA).

2.7. Statistical analysis

2.6. ELISA procedures

All alpacas infected with 1.1 × 103 , 1 × 104 or 1.2 × 105 viable C. pseudotuberculosis CFU, groups A, B and C, respectively, showed a febrile response (Fig. 1) with significant differences with respect to sham inoculated group D. In addition to changes in body temperature, swelling and abscess formation was observed at the inoculation site with positive isolation of C. pseudotuberculosis from pus that flowed through fistulae. After 2 weeks PI, the initial lesion progressed to scars of approximately one inch in diameter in all alpacas. Tumefaction of the draining left inguinal lymph nodes was observed as early as 1 week PI but alpacas did not show any behavior change and appetite was considered normal.

An indirect ELISA was carried out in polyestirene flatbottomed microtitre plates (Nunc Maxisorp F96, Roskilde, Denmark) as described (Ellis et al., 1990). Optimal dilutions of reagents were determined by block titrations. Negative control sera were obtained from an alpaca flock without a history of CLA, and were never raised with sheep. In addition, these alpacas did not present any lesions at slaughter, and samples of principal lymph nodes and viscera were negative for isolation of C. pseudotuberculosis. Positive control serum was obtained from an alpaca inoculated IM in both hind limbs with 0.219 mg/ml cell wall of C. pseudotuberculosis and Freund’s complete adjuvant (Difco laboratories BD Co., Maryland, USA). On day 35 booster injections were administered in the same way, but the antigen was mixed with Freund’s incomplete adjuvant (Difco laboratories BD Co., Maryland, USA). Antigen at 0.9 ␮g concentration in 50 ␮l of carbonate buffer pH 9.6 was dispensed in each odd well of the polyestirene plates. Control even wells contained only carbonate buffer pH 9.6. After incubation for 1 h at 37 ◦ C in a shaker, plates were washed 3 times with PBS-0.05% Tween 20. Plates were blocked with 100 ␮l of carbonate buffer with 3% skim milk was, except H-11 and 12 which were used as controls. After an incubation of 1 h at 37 ◦ C, plates were washed as above, and serum samples were dispensed at a dilution of 1/1000 in PBS plus 3% skim milk. After an additional incubation and washes, peroxidase labeled Protein A (Kirkergard & Perry laboratories, Gaithersburg-MD, USA) was diluted 1/500 in PBS plus 3% skim milk and used as conjugate. After 1 h of incubation and washes as above, 50 ␮l per well of TMB peroxidase solution (Kirkergard & Perry laboratories, Gaithersburg-MD, USA) was used as substrate. The color reaction was stopped with 2 M H2 SO4 . The optical density (OD) was read by use of a spectrophotometer set at 450 nm (Titertek multiskan II, Denmark). The cut off values for each antigen were determined as the corrected mean OD450 nm of the total number of serum samples from alpacas used as controls, plus 2 times the standard deviation.

The study was conducted as a completely randomized design in a factorial arrangement of treatments (groups). The model statements were day, treatment and the interaction day × treatment. Data were analyzed using the MIXED procedure of SAS (SAS, 1990) for the dependent variables, rectal temperature, and antibody optical density (OD). The experimental unit was the animal (n = 16) and the covariance structure selected was variance component. A probability difference lower than 0.05 (P < 0.05) was considered significant.

3. Results 3.1. Clinical responses to C. pseudotuberculosis primary infection

3.2. Clinical responses to C. pseudotuberculosis challenge After challenge exposure, the primary infected alpacas with C. pseudotuberculosis experienced a transient febrile response only during the first 24 h. In con-

Fig. 1. Least squares means estimates for body rectal temperature in Celsius (◦ C) for the alpacas infected at day 0 with 1 × 103 (A), 1 × 104 (B) or 1 × 106 (C) live C. pseudotuberculosis CFU, and sham inoculated group (D). The asterisk indicates level of significance: (*) P < 0.05; (**) P < 0.01; (***) P < 0.0001, respectively.

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Fig. 2. Least squares means estimates for body rectal temperature in Celsius (◦ C) for the alpacas challenged at day 0 (5 weeks post-primary infection of groups A, B and C, and sham inoculation of group D) with 9 × 108 live C. pseudotuberculosis CFU. The asterisk indicates level of significance: (*) P < 0.05); (**) P < 0.01); (***) P < 0.0001), respectively.

Fig. 3. Means estimates of optical densities by ELISA (450 nm) for the alpacas infected at week 0 with 1 × 103 (A), 1 × 104 (B) or 1 × 106 (C) live C. pseudotuberculosis CFU, and sham inoculated group (D). All alpacas were challenged with C. pseudotuberculosis 9 × 108 CFU at week 5 PI. The asterisk indicates level of significance: (*) P < 0.05; (**) P < 0.01; (***) P < 0.0001, respectively.

trast, the alpacas from the sham inoculated group D had a persistent elevated temperature with significant differences with respect to groups A, B and (Fig. 2).

were no significant differences among primary infected groups except for groups A and C at week 3 (P < 0.001). The sham inoculated alpacas (group D) did not have any response. After challenge exposure, the antibody response to C. pseudotuberculosis cell wall followed a weekly increasing trend with significant differences among the primary infected alpacas from groups A, B, C with respect to the na¨ıve group D at weeks 5, 6, 7 and 8 PI (Fig. 3). There were no significant differences among primary infected and challenged groups except for groups A–C and B–C at week 5 PI (P < 0.01 and P < 0.001, respectively).

3.3. Antibody responses after C. pseudotuberculosis primary infection and challenge The antibody response to the C. pseudotuberculosis cell wall followed a weekly increasing trend in primary infected alpacas with significant differences among infected alpacas from groups A, B, C, and the non infected group D at weeks 2, 3, and 4 (Fig. 3). There

Table 1 Location and number of abscesses (n) after primary inoculation with C. pseudotuberculosis (CFU) in the left flank of alpacas number 1, 2, 3 (group A); 4, 5, 6 (group B); 7, 8, 9 (group C); 10, 11, 12 (group D); 13, 14, 15 and 16 (group E), and subsequenttly challenged with 9 × 108 of viable bacteria in the right flank Treatment

A

(1.1 × 103 )

C. pseudotuberculosis

B (1 × 104 ) C. pseudotuberculosis

C (1.2 × 105 ) C. pseudotuberculosis

D PBS-sham inoculated

Alpaca #

Primary infection – left flank C. pseudotuberculosis

Challenge – right flank (9 × 108 ) C. pseudotuberculosis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Lr (2) Li Lr, Li Li Li, Lr (2) Lr Li, Mes (3) Lr (5) Li, Lr Li, Lr Li, Lr. Mes. (4) – – – – –

– – Ri – – – – – – – – – Ri,Rr Ria Ri, Mes. (4) Ri,Rr (3)

Symbols: (L) left; (R) right; (i) inguinal lymph node; (r) renal lymph node; (Mes) mesenteric Lymph node. a This alpaca died after 2 weeks of challenge.

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3.4. Gross pathology A necropsy exam was done on alpaca #13 from group D that suddenly died at day 17 after challenge inoculation. A subcutaneous edema was observed in the abdomen and perineal area, with a yellowish liquid that coagulated upon exposure to air. The regional lymphnodes from the prepuce and inguinal area were enlarged but without pus on cut surface. At the inoculation site on the right flank, a necrotic area of 5 cm, with purulent discharge, was observed. Internally, a 3 cm diameter abscess was observed in the renal lymph node. The distribution of body abscesses and bacterial isolation is summarized in Table 1. The primary infected alpacas had abscesses in the regional or internal renal-lymph nodes from the left body half, the primary inoculation side, with no lesions in the right challenged side except alpaca #3 from group A. This alpaca was primarily inoculated with low dose of C. pseudotuberculosis, and had an abscess in the regional lymph node from the right side. Three alpacas from group C inoculated with 1.2 × 105 C. pseudotuberculosis CFU, had regional (inguinal lymph node) and internal (renal lymph node) abscesses after primary infection; however, alpaca #12 from this group did not have any abscess after primary inoculation or after challenge exposure, except at the inoculation site. The primary sham inoculated alpacas had abscesses in the regional and internal lymph nodes from the right, challenged side. 4. Discussion Primary infections with low (1 × 103 ), moderate (1 × 104 ) or high (1 × 106 ) of live C. pseudotuberculosis CFU were protective against a challenge dose of 9 × 108 live C. pseudotuberculosis CFU, which produced a severe lymphadenitis in immune na¨ıve alpacas. Under natural conditions in the Andes, most of the abscesses in adult alpacas and llamas were observed externally in the mammary gland or were related with draining lymph nodes, and internally in the sublumbar area or renal lymph node (Braga, 1993). To corroborate the field observations of the lymphadenitis in adult alpacas we designed an infectious disease model by intradermal inoculation in the left flank with 1 × 106 CFU of C. pseudotuberculosis from alpaca origin trying to mimic a contaminated shearing wound (Braga, 1994). This model resembled the disease observed under natural conditions, with the presence of internal abscesses but not thoracic or lung abscesses as occurs in goats (Brown et al., 1985) or sheep (Brogden, 1984). In the present experiment we used lower doses of infection compared with the previous alpaca’s model,

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and with infective experiments in goats, with doses ranging from 1 × 105 to 1 × 109 CFU (Ashfaq and Campbell, 1980; Kuria et al., 2001). Our aim was to observe whether or not a primary low infection with C. pseudotuberculosis protects against a significant higher challenge dose. In other small ruminants like sheep and goats, and camels from the old world, the degree of protection induced by a primary infection varies according with the infective dose. In sheep, the inoculation of 1 × 107 CFU of streptomycin-sensitive C. pseudotuberculosis in the external left ear, protected against a 19R streptomycin resistant strain inoculated into the right external ear: The primary infected ewes did not develop lesions after challenge whereas, the immune na¨ıve ewes had multiple abscesses at the inoculation site, draining lymph nodes, internal lymph nodes and lungs (P´epin et al., 1993). In goats, small doses of 2 × 104 live C. pseudotuberculosis prevented the development of abscesses in draining or internal lymphnodes after a challenge dose of 4 × 106 C. pseudotuberculosis (Johnson et al., 1993). In camels from the old world, a primary infection of 5 × 109 CFU protected against a challenge after 3–5 months with 1 × 1010 CFU of C. pseudotuberculosis (Afzal et al., 1996). The present infective lower dose of 1.1 × 103 CFU of C. pseudotuberculosis used in alpacas was very small compared with previous references in other species like goats (Ashfaq and Campbell, 1980), sheep (P´epin et al., 1993) or old world camels (Afzal et al., 1996). We challenged the alpacas with a high dose of 9 × 108 C. pseudotuberculosis in the right flank and observed a severe disease characterized by fever, subcutaneous edema, and sudden death after 2 weeks of exposure in one of the immune na¨ıve alpacas, while the rest had a persistent fever and the presence of regional and internal abscesses. In consequence, the degree of protection induced by a primary infection in alpacas was not dose-dependant, since there were not significant differences among the groups with low, moderate or high dose of bacteria. On the other hand, the alpacas primarily infected with different low doses of C. pseudotuberculosis and subsequently exposed to a higher challenge dose, had a transient disease with fever only during the first 24 h with no abscess formation in the right body side that was inoculated with the challenge dose, except in one alpaca with a regional but with not internal abscesses. We observed that the degree of protection was not dependable of the dose, but with the presence of live bacteria. The protection could be affordable by the whole bacterium or its products like the toxin. In lambs the use of vaccines containing inactivated whole-cell antigen and detoxified exotoxin of C. pseudotuberculosis has been evaluated as vaccines reducing the prevalence of inter-

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nal abscesses (Tachedjian et al., 1995; Hodgson et al., 1999). A significant degree of protection was induced in mice vaccinated with a toxoid made from C. pseudotuberculosis, against a lethal challenge dose (Braga et al., unpublished data). It will be necessary to demonstrate the vaccine potential of different antigen composition from C. pseudotuberculosis to use in alpacas and llamas. We conclude that lower doses as small as 1.1 × 103 CFU of C. pseudotuberculosis in primary infected alpacas had a protective effect against a subsequent severe challenge exposure to this bacterium. Acknowledgments Especial thanks to Dr. James Higgins for statistical consultation and to Mrs. Jacque Staat and Mrs. Megan Haugh for the manuscript review. This work was supported by the International Foundation of Science IFS Stockholm Sweden, Grant B:2083-2 and the Consejo Nacional de Ciencia y Tecnologia CONCYTEC, Lima, Peru. References Afzal, M., Sakir, M., Majid, H.M., 1996. Corynebacterium pseudotuberculosis infection and lymphadenitis (Taloa or Mala) in the camel. Trop. Anim. Hlth. Prod. 28 (2), 158–162. Anderson, D.E., Rings, D.M., Kowalski, J., 2004. Infection with Corynebacterium pseudotuberculosis in five alpacas. J. Am. Vet. Med. Assoc. 11, 1743–1747. Ashfaq, M.K., Campbell, S.G., 1980. Experimentally induced caseous lymphadenitis in goats. Am. J. Vet. Res. 41, 1789–1792. Batey, R.G., 1986. Frequency and consequence of caseous lymphadenitis in sheep and lambs slaughtered at a Western Australian abattoir. Am. J. Vet. Res. 45, 557–561. Braga, W., 1993. Aislamiento de Corynebacterium pseudotuberculosis en alpacas y llamas adultas (Isolation of Corynebacterium pseudotuberculosis in adult alpacas and llamas). Revista de Investigaciones Pecuarias IVITA (Per´u) 6, 128–131. Braga, W. 1994. Corynebacterium pseudotuberculosis infection in alpacas: Pathogenesis and immunity. Thesis MS Universidad Nacional Mayor de San Marcos Lima, Peru. pp. 97. Brogden, K.A., 1984. Experimental Corynebacterium pseudotuberculosis infection in lambs. Am. J. Vet. Res. 45, 1532–1534. Brown, G.C., Olander, H.J., Biberstein, H., Moreno, D., 1985. Serologic response and lesions in goats experimentally infected with

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