Original research
Peer reviewed

Rapid application of long-acting ceftiofur can prevent death losses associated with Streptococcus equi subspecies zooepidemicus in pigs

Samantha J. Hau, DVM, PhD; Alexandra Buckley, DVM, PhD; Susan L. Brockmeier, DVM, PhD

Summary

Objective: Introduction of Streptococcus equi subspecies zooepidemicus strains into naive populations results in field mortality rates of 30% to 50% over 5 to 10 days. Because of the rapid disease progression, our goal was to determine whether antibiotic intervention could control S zooepidemicus disease in a group of animals following development of clinical signs.

Materials and methods: Thirty-two pigs were challenged with S equi subsp zooepidemicus. Following the development of clinical signs, 16 were treated with long-acting, injectable ceftiofur. Seven unchallenged pigs served as controls. Clinical signs were monitored following challenge and survival was compared between groups. Antibody titers were measured on day 0 and day 30 post challenge. On day 30 post challenge, 3 contact pigs were commingled with 2 treated animals to evaluate S equi subsp zooepidemicus transmission.

Results: Ceftiofur treatment eliminated clinical signs in 15 of 16 animals. However, multiple treatments were required to control disease in treated animals (2-3 doses providing 12-18 days of coverage). Antibody titers to S equi subsp zooepidemicus increased in challenged animals treated with ceftiofur, indicating sufficient exposure for immune stimulation. No contact pigs developed clinical signs of S equi subsp zooepidemicus following exposure.

Implication: Rapid application of injectable antibiotics is a viable method to reduce losses due to the introduction of S equi subsp zooepidemicus into a naive group of pigs and may help prevent transmission to contact animals following recovery.

Keywords: swine, Streptococcus equi subspecies zooepidemicus, septicemia, antibiotic

Received: December 21, 2021
Accepted: March 10, 2022

Resumen — La aplicación rápida de ceftiofur de acción prolongada puede prevenir pérdidas por muertes asociadas con Streptococcus equi subespecie zooepidemicus en cerdos

Objetivo: La introducción de cepas de Streptococcus equi subespecie zooepidemicus en poblaciones libres da como resultado tasas de mortalidad en el campo del 30% al 50% durante 5 a 10 días. Debido a la rápida evolución de la enfermedad, nuestro objetivo fue determinar si la intervención con antibióticos podría controlar la enfermedad por S zooepidemicus en un grupo de animales después del desarrollo de signos clínicos.

Materiales y métodos: Treinta y dos cerdos fueron desafiados con S equi subsp zooepidemicus. Tras el desarrollo de signos clínicos, 16 fueron tratados con ceftiofur inyectable de acción prolongada. Siete cerdos no desafiados sirvieron como controles. Los signos clínicos se monitorearon después de la exposición y se comparó la supervivencia entre los grupos. Los títulos de anticuerpos se midieron el día 0 y el día 30 después de la exposición. El día 30 después de la exposición, se mezclaron 3 cerdos contacto con 2 animales tratados para evaluar la transmisión de S equi subsp zooepidemicus.

Resultados: El tratamiento con ceftiofur eliminó los signos clínicos en 15 de 16 animales. Sin embargo, se requirieron múltiples tratamientos para controlar la enfermedad en los animales tratados (2-3 dosis que proporcionaron 12-18 días de cobertura). Los títulos de anticuerpos contra S equi subsp zooepidemicus aumentaron en animales desafiados tratados con ceftiofur, lo que indica una exposición suficiente para la estimulación inmunológica. Los cerdos sin contacto desarrollaron signos clínicos de S equi subsp zooepidemicus después de la exposición.

Implicación: La aplicación rápida de antibióticos inyectables es un método viable para reducir las pérdidas debidas a la introducción de S equi subsp zooepidemicus en un grupo de cerdos sin tratamiento previo y puede ayudar a prevenir la transmisión a animales contacto después de la recuperación.

Résumé — L’administration rapide de ceftiofur à action prolongée peut prévenir les mortalités associées à Streptococcus equi ssp zooepidemicus chez les porcs

Objectif: L’introduction de souches de Streptococcus equi ssp zooepidemicus dans des populations naïves entraîne des taux de mortalité sur le terrain de 30% à 50% sur 5 à 10 jours. En raison de la progression rapide de la maladie, notre objectif était de déterminer si l’administration d’antibiotique pouvait limiter la maladie à S equi ssp zooepidemicus dans un groupe d’animaux après le développement de signes cliniques.

Matériels et méthodes: Trente-deux porcs ont été infectés avec S equi ssp zooepidemicus. À la suite de l’apparition de signes cliniques, 16 ont été traités avec du ceftiofur injectable à action prolongée. Sept porcs non infectés ont servi de témoins. Les signes cliniques ont été surveillés après provocation et la survie a été comparée entre les groupes. Les titres d’anticorps ont été mesurés au jour 0 et au jour 30 après la provocation. Au jour 30 après l’infection, trois porcs contact ont été mélangés avec deux animaux traités pour évaluer la transmission de S equi ssp zooepidemicus.

Résultats: Le traitement au ceftiofur a éliminé les signes cliniques chez 15 des 16 animaux. Cependant, plusieurs traitements ont été nécessaires pour maitriser la maladie chez les animaux traités (2 à 3 doses fournissant 12 à 18 jours de couverture). Les titres d’anticorps contre S equi ssp zooepidemicus ont augmenté chez les animaux provoqués traités avec du ceftiofur, indiquant une exposition suffisante pour une stimulation immunitaire. Aucun porc contact n’a développé de signes cliniques d’infection à S equi ssp zooepidemicus à la suite de l’exposition.

Implication: L’administration rapide d’antibiotiques injectables est une méthode viable pour réduire les pertes dues à l’introduction de S equi ssp zooepidemicus dans un groupe de porcs naïfs et peut aider à prévenir la transmission aux animaux contact après la guérison.

Streptococcus equi subspecies zooepidemicus is a zoonotic pathogen that causes infections in a variety of mammalian species, including pigs.1-4 Historically, S equi subsp zooepidemicus infection has been sporadic in swine in the United States; however, in 2019, multiple introductions of a novel S equi subsp zooepidemicus strain occurred in the United States and resulted in mortality reaching 30% to 50% in affected groups of animals.3,4 Diseased animals had clinical signs including fever, severe lethargy, and reluctance to rise.3,4 Similar clinical signs were observed during experimental replication of disease, which progressed rapidly leading to 100% mortality 72 hours post infection.5

Currently, no in vivo studies have assessed the use of antimicrobials as an intervention strategy for S equi subsp zooepidemicus infection in pigs. Though S equi subsp zooepidemicus isolates, including the 2019 strains,3,4 are largely susceptible to β-lactam antibiotics,6,7 the rapid progression of disease could prevent antibiotic treatment from controlling losses associated with S equi subsp zooepidemicus introduction into a swine herd.

In this study, we investigated the use of long-acting ceftiofur crystalline free acid as an intervention strategy to control S equi subsp zooepidemicus infection in pigs. Our objective was to determine if rapid application of ceftiofur after the development of clinical signs could prevent losses associated with S equi subsp zooepidemicus infection.

Animal care and use

This animal study was approved by the US Department of Agriculture (USDA) Agricultural Research Service National Animal Disease Center Institutional Animal Care and Use Committee.

Materials and methods

Animal study

Thirty-nine, 10-week-old pigs were divided into three groups. Group 1 animals were challenged with S equi subsp zooepidemicus (n = 16). Group 2 animals were challenged with S equi subsp zooepidemicus and treated with a weight-calculated dose of ceftiofur crystalline free acid (Excede; Zoetis) given intramuscularly (n = 16). Group 3 animals were not challenged (n = 7). Animals in groups 1 and 2 were inoculated intranasally and orally with 3 mL of 2 × 108 colony forming units (CFU)/mL (1 mL per nostril and 1 mL orally). Animals in group 2 were treated with ceftiofur after the development of clinical signs on day 1 post challenge and again on day 5 post challenge when fevers started recurring. A third treatment was given only to animals developing a fever following retreatment. Body temperature was monitored twice daily following challenge by temperature chip (Destron Fearing). Following the challenge, animals were monitored every 4 hours excluding an 8-hour overnight period for the development of clinical signs including depression, reluctance to rise, and neurologic signs. Pigs were euthanized and necropsied if clinical signs became severe. At necropsy, the following samples were collected for culture: nasal swab, tonsil swab, serosal swab, liver swab, splenic swab, joint fluid, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid, and serum. Nasal and tonsil swabs were collected at 7- and 30-days post challenge in surviving animals to assess colonization.

On day 30 post challenge, 2 animals (692 and 697) that had been treated only twice with ceftiofur were commingled with 3 negative control animals from group 3 in a clean animal room to evaluate transmission from recovered animals to naive contacts following treatment. To allow time for development of serologic response, animals were monitored for clinical signs as previously described until day 63 when they were euthanized. At necropsy, samples were collected as previously indicated to screen for S equis subsp zooepidemicus.

Bacterial isolate and culture conditions

Streptococcus equi subsp zooepidemicus 19-031482-K1916623-LUNG1 (SRR10584760, https://www.ncbi.nlm.nih.gov/sra/SRR10584760) was isolated from a high mortality event in Tennessee in 2019.5,8 Streptococcus equi subsp zooepidemicus inoculum was grown on trypticase soy agar with 5% sheep blood (Becton Dickinson) at 37ºC with 5% CO2. Overnight S equi subsp zooepidemicus cultures were harvested in phosphate-buffered saline to an OD600 = 0.42, which results in 108 to 109 CFU/mL. The final concentration of S equi subsp zooepidemicus in the inoculum was quantified by plating serial dilutions. Animal samples were plated on trypticase soy agar with 5% sheep blood and incubated overnight at 37°C with 5% CO2 to assess for S equi subsp zooepidemicus. Suspect colonies were confirmed to be S equi subsp zooepidemicus by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) at the National Veterinary Services Laboratories in Ames, Iowa.

Serum antibody assessment

Serum was collected at the time of challenge and on day 30 post challenge from groups 1, 2, and 3. Serum was collected from contact pigs on day 0 (challenge), day 30 (commingling), and day 63 (approximately 4 weeks post commingling). All serum was stored at -80°C until enzyme-linked immunosorbent assays (ELISA) were performed.

For the ELISA, 96-well plates were coated with a 1:10 dilution of heat killed S equi subsp zooepidemicus (OD600 = 0.6) in carbonate-bicarbonate buffer. Titer was determined by serially diluting swine serum. Antibody was detected using horseradish peroxidase conjugated secondary antibody specific to the swine immunoglobulin heavy chain (1:20,000 dilution; SeraCare Life Sciences Inc) and tetramethylbenzidine substrate (Life Technologies). Optical density was measured at 450 nm with correction at 655 nm. Data were modeled with the nonlinear function of the log10 dilution and the log (agonist)-versus-response variable slope four-parameter logistic model in GraphPad Prism (GraphPad Software) with endpoint titer interpolated using two times the average reading for gnotobiotic swine serum.

Statistical analysis

Statistical analysis was completed in GraphPad Prism 8. Survival analysis was performed by the Kaplan and Meier product limit method comparing survival curves using the log-rank test. Antibody titers were converted to log10 values and compared using a two-way analysis of variance. A P value ≤ .05 was considered statistically significant.

Results

Clinical progression and survival

Challenge of pigs resulted in disease development by 24 hours post challenge (anorexia, fever, and depression) in group 1 and 2 animals. No clinical signs developed in nonchallenged animals (group 3). Following identification of clinical disease, group 2 animals were treated with a weight-calculated dose of ceftiofur. Most pigs (15 of 16) returned to normal behavior and normal body temperature by 24 hours post treatment (Figure 1). One animal (695) in group 2 developed neurologic signs (unable to rise, ataxic) following treatment and was euthanized 72 hours post challenge (Figure 2). Streptococcus equi subsp zooepidemicus was isolated from the CSF sample but absent from other collected samples. By day 5 post challenge (4 days post treatment), pigs were redeveloping clinical signs. They were treated a second time and temperatures and behavior returned to normal. A third treatment was required in 7 of 15 surviving animals in the treatment group as they became febrile at 10- to 11-days post challenge (Figure 1). Following the third treatment, animals did not develop further signs of disease, though temperature fluctuations were common (Figure 1).

Figure 1: Pig temperature following challenge with Streptococcus equi subsp zooepidemicus. Body temperature for each animal is plotted individually. Fever is indicated by the dashed line (40°C). All challenged animals developed a fever (> 40°C) following challenge. The challenged, treated pigs received treatment at 24 hours post challenge, which caused their temperatures to drop below 40°C. Temperatures rose again around 5 and 10 days post challenge and a second treatment of all pigs in the group was administered on day 5 post challenge, which brought temperatures back below 40°C. A third treatment was given only to pigs developing clinical signs or a fever (7 of 15 animals).
Figure

In contrast to group 2 animals, group 1 animals rapidly deteriorated without antibiotic intervention and 15 of 16 pigs were euthanized by 72 hours post challenge (Figure 2). Streptococcus equi subsp zooepidemicus was isolated from multiple systemic sites in all nontreated animals euthanized during the acute phase of infection.

Figure 2: Survival following a challenge with Streptococcus equi subspecies zooepidemicus. Most animals in the challenged group were euthanized by 72 hours post challenge (15 of 16). In contrast, most animals treated with ceftiofur recovered and only 1 animal was euthanized due to worsening clinical signs.
Figure 2

Nasal and tonsil swabs were taken on day 7 and day 30 post challenge to evaluate colonization. On day 7, no S equi subsp zooepidemicus was isolated from any of the surviving animals in group 2 (n = 15). Streptococcus equi subsp zooepidemicus was isolated from nasal swabs from the surviving nontreated animal (group 1, n = 1). By day 30 post challenge, S equi subsp zooepidemicus was not isolated from any of the surviving animals in group 1 (n = 1) or group 2 (n = 15).

Commingling with naive contact animals

On day 30 post challenge, 3 naive pigs from the nonchallenged controls (group 3) were commingled with 2 treated, challenged animals (group 2) that had only received 2 doses of ceftiofur. None of the commingled pigs showed behavior changes following commingling consistent with S equi subsp zooepidemicus disease; however, they developed a transient fever (> 40ºC; Figure 3), which lasted for 36 to 60 hours. By day 11 post commingling, all temperatures returned to normal and remained normal until the end of the study. At necropsy, nasal and tonsil swabs of contact animals were culture negative.

Figure 3: Body temperature following commingling of 2 challenged, treated pigs with 3 contact pigs. Body temperature for each animal is plotted individually. Fever is indicated by the dashed line (40°C). Transient fevers lasting 36-60 hours were noted in all 3 contact animals following commingling. All temperatures returned to normal by day 11 post commingling and remained normal for the rest of the study period.
Figure 3

Assessment of serum antibody titers

All groups had similar titers to S equi subsp zooepidemicus prior to challenge. At 30 days post challenge, a statistical increase in titer was seen in group 2 (15 of 16) and the surviving nontreated animal in group 1 (P < .001; Figure 4). The titer of treated animals was comparable to that of the surviving nontreated animal on day 30 (P = .47). No increase in titer was observed in the contact pigs following commingling (P = .07).

Figure 4: Antibody titers of pigs challenged with Streptococcus equi subsp zooepidemicus. Antibody titers were assessed on the day of challenge (D0) and the day of commingling (D30). Titers in the control pigs used as contacts were also measured on day 63 (33 days after commingling). An increased titer was noted in surviving challenged, treated animals (n = 15) and the surviving challenged animal (P < .001). The titer of commingled challenged, treated pigs on day 63 was comparable to control pigs on day 0 and day 30 (P = .74 and P = .07, respectively).
Figure 4

Discussion

In 2019, introduction of S equi subsp zooepidemicus into groups of naive swine in North America resulted in severe systemic disease and mortality reaching 30% to 50%.3,4 In this study, we evaluated antibiotic intervention to prevent severe death losses associated with S equi subsp zooepidemicus infection. We used injectable, long-acting ceftiofur, as the North American 2019 S equi subsp zooepidemicus isolates were found to be susceptible to β-lactam antibiotics.3,4

In nontreated animals, challenge with S equi subsp zooepidemicus produced clinical signs and mortality consistent with previous research.5 All challenged animals rapidly developed clinical disease and 15 of 16 nontreated animals were euthanized within 72 hours post challenge. However, treatment with ceftiofur following identification of clinical disease improved survival (15 of 16 animals survived) and clinical signs completely resolved for 3 days. Though retreatment was necessary, animals did stabilize by 15 days post challenge and no animal required more than 3 ceftiofur treatments (12-18 days of therapeutic plasma levels based on product information). Additionally, when evaluating antibody titers, treated pigs developed titers similar to the surviving nontreated pig by 30 days post challenge. This indicates that immune stimulation was sufficient to develop an adaptive immune response even with antibiotic treatment, and the antibiotic treatment was able to prolong survival until the immune response could develop.

One animal in the treated group developed neurologic signs and was euthanized following the first antibiotic treatment. Streptococcus equi subsp zooepidemicus was isolated from the animal’s CSF, but not from any of the other collected samples. This may be because S equi subsp zooepidemicus had already crossed the blood-brain barrier by the time of treatment, and the treatment was able to clear S equi subsp zooepidemicus from the systemic sites but not within the central nervous system. This indicates the importance of early detection of disease and rapid initiation of treatment to prevent S equi subsp zooepidemicus losses.

To assess whether treated pigs could serve as a reservoir for infection in naive pigs introduced following stabilization of S equi subsp zooepidemicus, we commingled 3 of the nonchallenged controls with 2 pigs that recovered following 2 treatments with ceftiofur. No contact animals developed anorexia or depression; however, all 3 developed a transient fever between day 1 and 11 post commingling that lasted 36 to 60 hours. Peak temperatures were not as high in contact pigs as were seen in primary challenged pigs (average 41.04°C versus 41.75°C, respectively), which could be due to a smaller exposure dose. Additionally, titers were evaluated to detect an exposure event. The absence of a rise in titers indicates any potential S equi subsp zooepidemicus exposure in the contact pigs did not stimulate an adaptive immune response. In other work, we have observed commingling of untreated pigs that survived an S equi subsp zooepidemicus challenge with naive contact pigs led to transmission in 2 of 3 contact animals, with one developing severe disease.9 Overall, the data from this study does not support a large exposure risk from previously infected, treated animals. In our experiment, the barn maintained high hygiene and animals were moved into a clean room for the commingling assessment, which minimized the potential for environmental exposure.

Overall, this study indicated that early treatment of S equi subsp zooepidemicus-infected animals with injectable ceftiofur can reduce clinical signs, reduce mortality, and provide time for the immune system to respond with an adaptive immune response. While we used ceftiofur, there is no reason to expect that treatment with other β-lactam antibiotics for the same duration would provide different efficacy. It is important to provide treatment rapidly after the detection of clinical signs. It is essential to provide treatment parenterally when clinical signs are present because acutely ill animals are off feed and do not drink, so antibiotics provided in feed or water would be ineffective.

Implications

Under the conditions of this study with an S equi subsp zooepidemicus challenge:

  • Long-acting ceftiofur reduced mortality.
  • Ceftiofur treatment protected animals without preventing an antibody response.
  • Exposure of naive pigs to recovered pigs did not result in disease.

Acknowledgments

The authors would like to thank the animal care staff for their assistance monitoring and handling the animals. This research was funded by the USDA. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer.

Conflict of interest

None reported.

Disclaimer

Scientific manuscripts published in the Journal of Swine Health and Production are peer reviewed. However, information on medications, feed, and management techniques may be specific to the research or commercial situation presented in the manuscript. It is the responsibility of the reader to use information responsibly and in accordance with the rules and regulations governing research or the practice of veterinary medicine in their country or region.

References

1. Timoney JF. The pathogenic equine streptococci. Vet Res. 2004;35(4):397-409. https://doi.org/10.1051/vetres:2004025

2. Baracco GJ. Infections caused by group C and G Streptococcus (Streptococcus dysgalactiae subsp equisimilis and others): Epidemiological and clinical aspects. Microbiol Spectr. 2019;7(2):e7.2.32. https://doi.org/10.1128/microbiolspec.GPP3-0016-2018

3. Sitthicharoenchai P, Derscheid R, Schwartz K, Macedo N, Sahin O, Chen X, Li G, Main R, Burrough E. Cases of high mortality in cull sows and feeder pigs associated with Streptococcus equi subsp zooepidemicus septicemia. J Vet Diagn Invest. 2020;32(4):565-571. https://doi.org/10.1177/1040638720927669

4. de O Costa M, Lage B. Streptococcus equi subspecies zooepidemicus and sudden deaths in swine, Canada. Emerg Infect Dis. 2020;26(10):2522-2524. https://doi.org/10.3201/eid2610.191485

5. Hau SJ, Lantz K, Stuart KL, Sitthicharoenchai P, Macedo N, Dersheid RJ, Burrough ER, Robbe-Austerman S, Brockmeier SL. Replication of Streptococcus equi subspecies zooepidemicus infection in swine. Vet Microbiol. 2022:264:109271. https://doi.org/10.1016/j.vetmic.2021.109271

6. Awosile BB, Heider LC, Saab ME, McClure JT. Antimicrobial resistance in bacteria isolated from horses from the Atlantic Provinces, Canada (1994 to 2013). Can Vet J. 2018;59(9):951-957.

7. Erol E, Locke SJ, Donahoe JK, Mackin MA, Carter CN. Beta-hemolytic Streptococcus spp. from horses: A retrospective study (2000-2010). J Vet Diagn Invest. 2012;24(1):142-147. https://doi.org/10.1177/1040638711434138

8. Chen X, Resende-De-Macedo N, Sitthicharoenchai P, Sahin O, Burrough E, Clavijo M, Dersheid R, Schwartz K, Lantz K, Robbe-Austerman S, Main R, Li G. Genetic characterization of Streptococcus equi subspecies zooepidemicus associated with high swine mortality in the United States. Transbound Emerg Dis. 2020;67(6):2797-2808. https://doi.org/10.1111/tbed.13645

9. Hau SJ, Buckley A, Brockmeier SL. Bacterin vaccination provides insufficient protection against Streptococcus equi subspecies zooepidemicus infection in pigs. Front Vet Sci. 2022;9:827082. https://doi.org/10.3389/fvets.2022.827082

National Animal Disease Center, Agricultural Research Service, US Department of Agriculture, Ames, Iowa.

Corresponding Author: Dr Samantha J. Hau, 1920 Dayton Ave, Ames, IA 50010; Tel: 515-337-6260; Email: Samantha.Hau@usda.gov.

Hau SJ, Buckley A, Brockmeier SL. Rapid application of long-acting ceftiofur can prevent death losses associated with Streptococcus equi subspecies zooepidemicus in pigs. J Swine Health Prod. 2022;30(5):292-297. https://doi.org/10.54846/jshap/1298

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