HORSES

Antimicrobial susceptibility pattern of selected equine pathogens from clinical cases submitted to the AHL between May 2007 and May 2015

Durda Slavic, Murray Hazlett, Beverly McEwen, Michael Deane, Melanie Barham

The use of laboratory testing (culture and antimicrobial susceptibility panels), plus prudent counselling of owners, can assist in stewardship of antimicrobial use. To assist decision-making, we searched the AHL laboratory information system for the susceptibility patterns of common equine pathogens for the past 8 years (Fig. 1). Results were available for 155 Actinobacillus equuli (A. equuli subsp. equuli and A. equuli subsp. haemolyticus), 382 Escherichia coli, 792 Streptococcus equi subsp. zooepidemicus, and 131 Streptococcus equi subsp. equi isolates.

There was a high level of resistance in beta-haemolytic Streptococcus spp. to aminoglycosides, including amikacin and kanamycin, and a lower level of resistance to gentamicin. As expected, Streptococcus spp. isolates were sensitive to penicillin, which should be a primary drug of choice for their treatment. The susceptibility patterns for E. coli and A. equuli were less predictable, with resistance being present to all drugs used for testing but at different levels (Fig. 1). Note that E. coli is intrinsically resistant to penicillin, therefore this antimicrobial is not used for testing of E. coli isolates.

When extrapolating from laboratory data to field cases from the information shown in Fig. 1, one should exercise caution. Laboratory data in general are biased in that they are often associated with clinical cases that may have undergone previous treatments. For that reason, the level of resistance in our data may be higher than in the general population. In addition, from the clinical perspective, our data are based on in vitro results. These results may or may not be reproduced in vivo.

Veterinarians are in the front-line in the war against antimicrobial resistance, and in maintaining the efficacy of antimicrobials, and as such need to avoid the use of category I antimicrobials, such as third (ceftiofur) and fourth generation cephalosporins, and fluoroquinolones (enrofloxacin), and limit use of category II drugs, e.g., aminoglycosides, the first and second generations of cephalosporins including cephamycins, penicillins, and trimethoprim/sulfamethox-azole - these 2 categories include most of the antimicrobials used in equine practice (see next page). Serum amyloid A is being used by many as a marker/decision factor in determining antimicrobial use in horses, and is included in all equine biochemical panels at the AHL.   

 

Figure 1. Antimicrobial resistance patterns of selected   equine pathogens from 2007 to 2015. Data are shown as % resistant.

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Categories of antimicrobials used in equine practice, as they relate to human medical treatment.

Adapted from Health Canada’s categorization of antimicrobials.

1. Category I: Very High Importance

These antimicrobials are considered of very high importance in human medicine as they meet the criteria of being essential for the treatment of serious bacterial infections and limited or no availability of alternative antimicrobials for effective treatment in case of emergence of resistance to these agents. Examples include:

· Carbapenems, e.g., imipenem

· Cephalosporins, third and fourth generations, e.g., ceftiofur

· Fluoroquinolones, e.g., ciprofloxacin, enrofloxacin

· Glycopeptides, e.g., vancomycin

· Nitroimidazoles, e.g., metronidazole

· Penicillin-β-lactamase inhibitor combinations, e.g. amoxicillin-clavulanic acid

· Polymyxins (colistin), e.g. polymyxin B

· Therapeutic agents for tuberculosis, e.g. rifampin

2. Category II: High Importance

Antimicrobials in this category consist of those that can be used to treat a variety of infections including serious infections and for which alternatives are generally available. Examples include:

· Aminoglycosides (except topical agents), e.g., gentamicin, amikacin, kanamycin

· Cephalosporins, first and second generations (including cephamycins), e.g., cefazolin

· Macrolides, e.g., clarithromycin, erythromycin, azithromycin

· Penicillins, e.g., penicillin, amoxicillin, ampicillin

· Quinolones (except fluoroquinolones), e.g., nalidixic acid

· Trimethoprim/sulfamethoxazole (TMS)

3. Category III: Medium Importance

Examples include:

· Aminoglycosides, topical agents

· Bacitracins, e.g., BNP eye ointment

· Nitrofurans, e.g., nitrofurazone

· Phenicols, e.g., chloramphenicol

· Sulfonamides

· Tetracyclines, e.g., doxycycline, oxytetracycline, minocycline

· Trimethoprim

4. Category IV: Low Importance

Antimicrobials in this category are currently not used in human medicine. These drugs are not frequently used in equine medicine either.

 

Reference   Health Canada, Categorization of Antimicrobial Drugs Based on Importance in Human Medicine.

http://www.hc-sc.gc.ca/dhp-mps/vet/antimicrob/amr_ram_hum-med-rev-eng.php

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Equine granulocytic anaplasmosis (Anaplasma phagocytophilum)

Kristiina Ruotsalo, Brent Hoff, Richard Ryan

A 17-year-old Appaloosa mare with a history of long-term oral prednisone treatment for severe recurrent airway obstruction (RAO), was presented to the referring veterinarian with a recent history of anorexia, depression, reluctance to move, and limb and ventral edema. Clinical evaluation revealed jaundice, pyrexia (40.5^oC), tachypnea, and tachycardia. A week previously, this mare had been treated for mild colic, which had resolved.

EDTA blood and serum were submitted to the AHL for a comprehensive CBC and biochemistry panel, and intravenous treatment with trimethoprim/sulfadiazine, flunixin, and Newcells was started while awaiting laboratory results.

The CBC demonstrated marked thrombocytopenia (platelet count 28 X10⁹ /L), and a mild left shift (0.40 X10⁹/L). Examination of the peripheral blood smear revealed that ~35% of neutrophils contained intracytoplasmic morulae consistent with Anaplasma phagocytophilum (Figures 1, 2). PCR subsequently confirmed the identity of these organisms as A. phagocytophilum.

Significant changes in the serum biochemistry profile included mild to moderate hypoproteinemia (total protein 48 g/L), a moderate increase in free bilirubin (142 µmol/L), and a marked increase in serum amyloid A (2,668 mg/L).

Following definitive identification of A. phagocytophilum, treatment was changed to intravenous oxytetracycline, administered once daily for 5 days. The mare responded well to treatment, and by completion of the antibiotic regime was noticeably less jaundiced with almost complete resolution of limb edema. A serum biochemistry profile and CBC taken 14 days from the date of presentation  revealed a total serum protein of 58 g/L, free bilirubin of 5 µmol /L, and serum amyloid A of 0 mg/L. The platelet count was 175 X 10⁹/L, no left shift was noted, and no morulae could be visualized within the neutrophils. A. phagocytophilum PCR was negative. The mare was clinically normal according to the owner.

A. phagocytophilum includes the organisms previously described as Ehrlichia equi, Ehrlichia phagocytophila, and the human granulocytic ehrlichiosis agent, to reflect close genome homology. A. phagocytophilum is the causative agent of equine granulocytic anaplasmosis (EGA), and is found in membrane-bound vacuoles within the cytoplasm of infected neutrophils and eosinophils. These inclusion bodies consist of one or more coccoid or coccobacillary organisms ~0.2 µm in diameter, as well as large, granular aggregates (morulae), visible under light microscopy.

EGA typically occurs during late fall, winter, and spring. Horses of any age are susceptible, but clinical signs are less severe in horses <4 years of age. The disease is not contagious but thought to be transmitted by the western black-legged tick (Ixodes pacificus) in western North America, and the deer tick (Ixodes scapularis) in eastern North America. The pathogenesis of EGA is poorly understood, but after entering the dermis by tick-bite inoculation, the bacteria invade target cells of the hematopoietic and lymphoreticular systems. It is unclear if there is direct injury of infected cells, but localized inflammatory events are initiated within tissues containing infected cells. Peripheral sequestration, consumption, and destruction of peripheral blood components are all proposed as potential mechanisms of cytopenias.

Following an incubation period of ~10 days, infected horses may experience subclinical disease or develop overt clinical signs including fever, depression, anorexia, reluctance to move, limb edema, icterus, petechiae, and ataxia. Moderate to severe morbidity is seen occasionally with EGA, and occasional mortality has been reported. The disease is often self-limiting, and clinical signs usually last 7-14 days. Anemia, variable leukopenia and thrombocytopenia are usually noted with clinical cases of EGA.

Diagnosis relies upon clinical awareness of geographic areas for infection, appropriate clinical signs and laboratory changes, and the presence of morulae within granulocytes in peripheral blood smears. As affected horses may be leukopenic, and because the number of granulocytes containing morulae can vary from 1% to 50% by day 3-5 of infection, examination of a buffy coat smear can enhance detection of morulae. PCR can confirm the clinical diagnosis and is particularly helpful in both the early and late stages of disease when the numbers of organisms may be too small for diagnosis by microscopy. PCR testing for A. phagocytophilum is available at the AHL, and can be used for detection of the organism from whole blood, tissue, and ticks.

A .phagocytophilum has not been reported previously in Ontario. Prevalence of this disease in Ontario is unknown, but anaplasmosis should be considered as a differential diagnosis in horses exhibiting appropriate clinical signs, particularly unexplained peripheral cytopenias. Areas of concern are the north shore of Lake Ontario, northwestern Ontario along the Manitoba/Minnesota border, and possibly the north shore of Lake Erie.   

Figure 1. A. phagocytophilum morula in a neutrophil.	Figure 2. Two A. phagocytophilum morulae in a neutrophil.