Diphtheria Toxoid

History of the Disease

Historical descriptions of diphtheria-like illness (throat membrane, neck swelling, frequent suffocation) first appeared in ancient Egyptian writings from the second millennium bc with increasingly detailed descriptions by Greek writers, including Hippocrates (fifth century bc ), Aretaeus (second century ad ), and Aëtius (sixth century ad ). Only isolated reports of the disease appeared until the 17th century, when devastating outbreaks occurred in Spain: 1613 was known as the Year of Strangulations (Año de los Garrotillos). ^, In the second half of the 19th century, increasingly severe epidemics swept many parts of Europe and large cities in the United States. ^{, ,}

Due to these epidemics, vigorous efforts were made by researchers in the new field of bacteriology to identify the causative agent. In the early 19th century, Bretonneau clearly delineated the clinical picture of diphtheria, convincingly argued for its communicability, successfully pioneered tracheostomy as a method of treatment, and gave the disease its name, derived from the Greek word for leather or tanned skin. ^, In 1883, Klebs first described the characteristic organisms in stained preparations of diphtheritic membranes, and Löffler reported the successful growth of these organisms in culture a year later. Shortly thereafter, Roux and Yersin demonstrated the presence of a potent exotoxin (see “Passive Immunization”). Over the next decade, antisera produced in animals by injection of sublethal or inactivated cultures were first shown to prevent death in nonimmune animals that were challenged with virulent organisms, and then to prevent death in children with clinical diphtheria. ^,

The concept of active immunization began with Theobald Smith in 1907, who noted that long-lasting immunity to diphtheria could be produced in guinea pigs by the injection of mixtures of diphtheria toxin and antitoxin and suggested that these mixtures might do the same for humans. After successful immunization of children by von Behring with toxin–antitoxin mixtures, immunization programs began in selected European and American cities. ^, In the early 1920s, Ramon showed that diphtheria toxin, when treated with heat and formalin, lost its toxic properties but retained its ability to produce serologic protection against the disease. Thus the current immunizing preparation, diphtheria toxoid, came into being.

Why the Disease Is Important

Before the introduction of diphtheria immunization, diphtheria was a major cause of childhood mortality, and it remains endemic in many developing countries. While the disease has disappeared in many parts of the world, persistent outbreaks, especially in regions with economic decline, civil unrest, and population displacement, have highlighted the ability of this ancient scourge to re-emerge among populations that fail to maintain protective immunity through vaccination.

Clinical Description

Classic respiratory diphtheria has an insidious onset after an incubation period of typically 1–5 days (range: 1–10 days). Symptoms are initially nons-pecific and mild. At the very onset of symptoms, no pharyngeal membrane may be present. About a day after onset, small patches of exudate appear in the pharynx; within 2 or 3 days, these patches spread and become confluent and may form a membrane that covers the entire pharynx, including the tonsillar areas, soft palate, and uvula. This membrane becomes grayish, thick, and firmly adherent to the underlying mucosa. Efforts to dislodge the membrane typically result in bleeding. Anterior cervical lymph nodes become markedly enlarged and tender. In a proportion of patients, the lymph node swelling is associated with considerable inflammation and edema of the surrounding soft tissues, giving rise to the so-called bullneck appearance, which is associated with a higher morbidity and mortality. Untreated patients characteristically appear toxic until the membrane softens and detaches about a week after onset; acute symptoms dissipate during this time.

Although pharyngeal diphtheria is by far the most common form of disease seen in unimmunized populations, other sites may be involved. Laryngeal diphtheria occurs in 25% of cases; in 75% of these instances, the pharynx is also involved. Laryngeal diphtheria may occur at any age, but is particularly likely to occur in children younger than 4 years. It is marked by an insidious onset with gradually increasing hoarseness and stridor. Laryngeal diphtheria is associated with greater morbidity and mortality as a result of airway obstruction and the greater degree of toxin absorption from the extensive membrane. Isolated nasal diphtheria is uncommon (approximately 2% of cases). Cutaneous, aural, vaginal, and conjunctival diphtheria together account for only approximately 2% of cases and are often secondary to nasopharyngeal infection.

Cutaneous diphtheria caused by toxigenic C. diphtheriae is an indolent skin infection that often occurs at sites of burns or other wounds and may act as a source of respiratory infection. Systemic complications are uncommon with cutaneous diphtheria. Historically it was believed to be more common in warmer climates and in poor social conditions; however, cases have been reported in less temperate locations, typically after travel from a diphtheria-endemic area. ^,

Invasive disease caused by C. diphtheriae can occur, most commonly as a result of nontoxigenic strains, with bacteremia, endocarditis, osteomyelitis, and arthritis reported. Disease can also be caused by toxin-producing strains of two other Corynebacterium species: C. ulcerans and C. pseudotuberculosis . Both species are zoonotic; infections have been documented in pigs, cattle, dogs, and cats. In humans, toxin-producing C. ulcerans may cause respiratory or cutaneous illness indistinguishable from that caused by toxigenic C. diphtheriae , while C. pseudotuberculosis may cause lymphadenitis. ^,

Complications

The impact of diphtheria is largely measured by complications attributable to the local disease and to the effect of absorbed toxin on other organs. The major threat from laryngeal diphtheria is respiratory obstruction. The membrane may extend down into the tracheobronchial tree, resulting in pneumonia and expiratory respiratory obstruction. Because of edema of the upper respiratory tract, pharyngeal and nasal diphtheria are associated with secondary otitis media and sinusitis.

The majority of deaths from diphtheria result from the effects of absorbed diphtheria toxin on various organs; severe complications from toxin absorption include acute systemic toxicity, myocarditis, and neurologic complications, primarily peripheral neuritis. The risk for complications is directly proportional to the extent of local disease, presumably because of increased production and absorption of the toxin in larger membranes. In the past it was believed that disease severity could be related to the strain biotype (gravis, intermedius, belfanti, and mitis), ^, however, this has been disproven.

Severe acute systemic toxicity with myocardial involvement can occur between the third and seventh day of the illness; this so-called early myocarditis is usually fatal. Late myocarditis usually appears in the second or third week of illness, when the local symptoms of diphtheria in the respiratory tract are resolving and the patient is otherwise improving.

In either early or late myocarditis, a wide variety of clinical and electrocardiographic findings may be noted. Electrocardiography most often shows conduction changes and alterations in T waves; supraventricular and ventricular ectopic rhythms are common in severe diphtheria. The earlier electrocardiographic changes appear, the worse the prognosis. Complete heart block frequently occurs and is usually fatal; ventricular pacing may not improve survival. Echocardiograms show decreased contractility and ventricular dilation proportional to the severity of the clinical carditis; a left ventricular ejection fraction of less than 35% is associated with an increased risk for death. ^, Although electrocardiograms and echocardiograms return to normal in most survivors, residual changes are seen in some survivors of severe carditis for up to several years after illness. ^{, ,} Left bundle-branch block at hospital discharge has a poor prognosis.

Neurologic complications of diphtheria are primarily toxic peripheral neuropathies, and they occur in approximately 15–20% of cases. ^, The manifestations are more motor than sensory and usually begin 2–8 weeks after onset of the illness. In severe cases, palatal paralysis with consequent nasal voice and nasal regurgitation of ingested fluids may occur during the acute membranous phase, particularly with extensive pharyngeal disease, and are believed to be attributable to local effects of the toxin. With milder disease, palatal paralysis is common as late as the third week. Symmetric peripheral neuritis of the lower extremities is a frequent neurologic complication, usually occurring 3–10 weeks after onset of the infection. Diaphragmatic paralysis occasionally occurs, usually a month or more after onset, and may require mechanical respiratory support. Ocular paralysis, involving either the extraocular muscles or those of accommodation, sometimes appears, usually 5 or 6 weeks after onset. Fortunately, functional recovery from these neuropathies is the rule, even in severe disease. An association between diphtheria and delayed-onset hearing loss has also been reported.

Case fatality from respiratory diphtheria infection prior to the advent of diphtheria antitoxin treatment and vaccination was thought to be on the order of 40–50%, although data are limited. In the current era, case fatality ranges from 5% to 10%, although outbreaks in resource-limited settings note a wider rate range, from 3% to 33%. ^,

Bacteriology

Corynebacterium diphtheriae is a slender, Gram-positive bacillus, usually with one end being wider, thus giving the often-described club-shaped appearance. On culture, particularly under suboptimal conditions, characteristic bands or granules appear. On smear, the organisms often have a “pick-up sticks” relationship, assuming parallel (palisade-like), or V- or L-type patterns. The organisms are resistant to environmental changes, such as freezing and drying. There are four biotypes of C. diphtheriae (gravis, mitis, belfanti, and intermedius), which historically were identified by colonial morphology and biochemical differences; however, in practice, only the intermedius biotype can be distinguished reliably by colonial morphology.

Pathogenesis as It Relates to Prevention

The exotoxin produced by C. diphtheriae is by far the most important pathogenic factor associated with the organism. The ability of C. diphtheriae strains to produce toxin results from a nonlytic infection by bacteriophages (β-corynebacteriophage) that contain a genetic sequence encoding the toxin. The phage integrates into specific sites present in C. diphtheriae and other Corynebacterium species. The presence of the phage is thought to confer a survival advantage to the bacterium by increasing the probability of transmission in a susceptible population; transmission may be facilitated by local tissue damage resulting from the toxin. ^, The sequence of diphtheria toxin is highly conserved in C. diphtheriae strains, suggesting that immunologically important differences among the toxins produced by different strains are unlikely to occur. Once integrated, the tox gene is part of a multiple bacterial gene operon; other bacterial gene products in this operon are involved in the liberation and uptake of host iron. The entire operon is under the control of a repressor gene, dtxR , which in the presence of iron binds to and inhibits the tox gene; therefore, toxin is produced only under low-iron conditions.

Diphtheria toxin is a polypeptide with a molecular weight of approximately 58,000 Da. The toxin is secreted as a proenzyme, requiring enzymatic cleavage into two fragments (fragments A and B) to become active. Fragment B is responsible for attachment to and penetration of the host cell. Although nontoxic by itself, fragment B appears to be the antigen responsible for immunity. The receptor domain of fragment B binds to a cell surface receptor, heparin-binding epidermal growth factor precursor, with CD9 as a coreceptor. After receptor-mediated endocytosis, fragments A and B are detached. The released fragment A is the toxic moiety, and it acts by inhibiting protein synthesis, resulting in cell death. Unless cell penetration occurs, fragment A is inactive. Differences in the tissue distribution of the receptor and coreceptors may account for the differential effects of diphtheria toxin on different organs. ^,

The ability of tox gene–containing bacteriophages to infect nontoxigenic strains of C. diphtheriae provides a potential explanation for the fact that, during outbreaks of diphtheria, both toxin-producing and non–toxin-producing strains of the organism may be isolated on culture surveys. Some evidence suggests that the introduction of a toxin-producing strain of C. diphtheriae into a community may occasionally initiate an outbreak by transfer of phage to nontoxigenic strains of the organism carried in the respiratory tracts of community inhabitants, rather than a new strain being the responsible agent.

On mucous membranes, the toxin causes local cellular destruction, and the accumulated debris and fibrin result in the characteristic membrane. More important, absorbed toxin is responsible for remote manifestations affecting various organs, including the myocardium and nervous system. Because the lethality of diphtheria is almost entirely determined by the organism’s toxin, clinical immunity depends primarily on the presence of antibodies to the toxin.

Two other closely related Corynebacterium species, C. ulcerans and C. pseudotuberculosis , have been demonstrated to produce diphtheria toxin. Similarly to C. diphtheriae , nontoxigenic C. ulcerans and C. pseudotuberculosis strains can be converted to toxigenic strains by infection with β-corynebacteriophage.

Modes of Transmission

Transmission is from person to person, by respiratory droplet. Physical contact with cutaneous lesions also appear to be important in transmission under poor or crowded social conditions. ^{, ,} In addition, evidence of outbreaks caused by contaminated milk and milk products has been reported. ^, The organism is reasonably hardy and has been isolated from the environment of persons infected with C. diphtheriae . Nonetheless, the occurrence of indirect transmission by contact with dust or fomites has not been established.

Diagnosis

Diphtheria is rare in the United States, however, some developing countries continue to have high levels of circulation of toxigenic strains of C. diphtheriae , and persistent strains have also been reported in some highly developed countries. Healthcare providers should be aware of the signs and symptoms that suggest diphtheria, as respiratory and cutaneous diphtheria cases have been reported following travel to endemic areas.

The hallmark of respiratory diphtheria is the presence of pseudomembrane in the pharynx. In countries where the disease is endemic, a patient with confluent pharyngeal exudate should be suspected of having diphtheria until proven otherwise. Laryngeal diphtheria usually occurs concomitantly with pharyngeal involvement, therefore membranous pharyngitis with stridor also should be considered to be diphtheria until proved otherwise. The differential diagnosis includes epiglottitis caused by Hemophilus influenzae type b (Hib), although this is uncommon in immunized populations, spasmodic croup, the presence of a foreign body, or viral laryngotracheobronchitis.

Nasal diphtheria may be difficult to distinguish from many other causes of nasal discharge and accordingly is most likely to be suspected if the patient has been exposed to diphtheria, such as during an outbreak. Suspicion should be heightened if a serosanguineous discharge is present and if the upper lip is ulcerated. Any cutaneous or mucous membrane lesions at other sites should be considered suspicious if a membrane is noted.

Swabs for culture should be obtained under direct visualization, preferably from the edge or beneath the edge of the membrane. Directly stained smears are usually grossly misleading even in experienced hands and should not be used. Swabs should be inoculated promptly onto tellurite-containing media and onto blood agar. Cultures should be incubated promptly and interpreted by an experienced microbiologist. Because not all C. diphtheriae recovered on culture are toxigenic, testing for toxin production must be performed. The modified Elek immunoprecipitation test for detection of toxin is the standard assay, but it generally requires 24–48 hours. Many laboratories now use a polymerase chain reaction (PCR) assay for the detection of tox gene. which can provide a rapid indication that an isolate may be toxigenic, and can be performed directly on clinical specimens. Real-time PCR assays that detect tox gene also have been developed. Because in some isolates the tox gene is present but nonfunctional, toxin production should always be confirmed by an immunoprecipitation test.

Typing of strains of C. diphtheriae has been used as an adjunct to epidemiologic investigations. Recent approaches include ribotyping, multilocus sequence typing, and whole genome sequencing. Historically, ribotyping has been used extensively and standard nomenclature has been developed. Multilocus sequence typing also has been used and appears useful for discriminating reliably among strains of C. diphtheriae . A combination of these two methods in addition to other molecular typing have been used to characterize the dominant strains of C. diphtheriae associated with the outbreak in the former Soviet Union, and to document ongoing endemic circulation in certain communities in the United States and Canada. ^{, ,} As whole genome sequencing has become more accessible, this method is being used with increased frequency to discriminate among C. diphtheriae strains, evaluate its genomic diversity, and identify infection sources and transmission patterns. ^,

Rapid diagnostic tests would be an extremely useful adjunct to currently available testing, especially in resource-limited settings where outbreaks are apt to occur. While a highly accurate immunochromatographic strip test that detects diphtheria toxin was developed in the early 2000s, it is not widely available.

Treatment

The risk of complications and mortality from diphtheria are inversely related to the promptness of treatment. Thus, it is critical that when diphtheria is suspected, treatment should be initiated immediately after bacteriologic specimens are obtained, without waiting for results.

Equine antitoxin is the gold standard treatment for diphtheria, and is given intramuscularly or intravenously; many authorities prefer the intravenous route because a therapeutic blood level can be reached more rapidly. The entire therapeutic dose should be administered at one time, and the amount of antitoxin recommended varies between 20,000 and 100,000 units. Larger amounts are recommended for persons with extensive local lesions, because the amount of toxin produced depends on the size of the membrane. Toxin that has already entered host cells is unaffected by antitoxin. In the United States, antitoxin is available through an U.S. Food and Drug Administration (FDA) Investigational New Drug protocol and may be requested through Centers for Disease Control and Prevention (CDC) by calling 770-488-7100.

Although diphtheria antitoxin is the mainstay of therapy, penicillin or erythromycin should be given to hasten clearance of the organism, prevent transmission, and cease further production of diphtheria toxin. ^, Azithromycin has also been utilized for treatment in outbreaks, although data on its effectiveness are limited to in vitro studies. Antimicrobial treatment with penicillin or erythromycin should be continued for 2 weeks. On completion of treatment, patients should be cultured twice at least 24 hours apart to confirm elimination C. diphtheriae . Patients who continue to harbor the organism after antibiotic treatment should receive an additional 10-day course of oral erythromycin, and specimens for follow-up cultures should be obtained on completion of the course. ^, Although antimicrobial treatment has no apparent effect on the clinical course of the disease, in most instances the organism can no longer be recovered on culture within a week of therapy and subsequent convalescent carriage is thus uncommon.

Epidemiology in the United States

Occurrence of respiratory diphtheria in the United States has decreased dramatically from 147,000 cases in 1920 to an annual average of <1 reported cases from 1996 through 2019 ( Fig. 21.1 ). Use of diphtheria antitoxin in the 1920s, in addition to the introduction of diphtheria toxoid vaccination programs in the 1940s, played a large role in this steep decline. By the late 1950s, the incidence of diphtheria was markedly reduced in the United States, but disease continued to affect some areas and populations. From 1959 to 1970, 5048 cases of diphtheria were reported in the United States; the highest incidence rates were reported in the southeast, south central, and northern plains states, and there was disproportionately higher incidence among American Indian and Black persons than among White persons. From 1971 to 1981, the number of cases reported declined further (853 noncutaneous cases), however geographic and racial and ethnic disparities continued, with incidence rates exceeding 1 per million population in South Dakota, New Mexico, Alaska, Washington, Arizona, and Montana, and rates 100-fold greater for American Indian persons than for White or Black persons.

From 1980 to 1995, only 41 cases of respiratory diphtheria were reported in the United States, and even fewer were reported from 1996 to 2018 (14 cases). Two respiratory cases have been reported since 2012, one in 2014 and one in 2018; the disease in both instances was caused by nontoxigenic C. diphtheriae and both patients were fully vaccinated. In comparison, the last nonimported case of respiratory disease caused by toxigenic C. diphtheriae was reported in 1997. It is important to note that the U.S. reporting case definition from 1980 to 2018 did not require confirmation of toxin production, and therefore cases reported during this time included respiratory disease caused by both toxigenic and nontoxigenic C. diphtheriae .

While cutaneous diphtheria was not recognized by the U.S. case definition from 1980 to 2018, it has been increasingly detected. Cutaneous outbreaks were reported in the past, with the last occurring in the 1970s among residents of Skid Road in Seattle, Washington. More recently reported cutaneous cases have been sporadic, often following travel to diphtheria endemic zones; four imported cases of cutaneous toxigenic C. diphtheriae were detected from 2014 to 2018. Given that cutaneous toxigenic disease can be transmitted and cause respiratory disease, the U.S. reporting case definition was revised in 2019 to include disease caused by toxigenic C. diphtheriae from any site. There were two toxigenic diphtheria cases reported in 2019 in the United States; both were nonrespiratory and associated with travel.

Toxigenic C. ulcerans is infrequently detected in the United States; from 1996 to 2019, six cases were identified, five with respiratory disease and one with conjunctivitis. ^, The disease was fatal in two of the six cases although they did not receive diphtheria antitoxin.

Despite the low number of diphtheria cases reported in the United States, it is possible that C. diphtheriae continues to circulate among undervaccinated populations. While little disease was reported in the 1990s, surveillance revealed widespread circulation of the organism in one American Indian community in the Northern Plains in 1996. Similarly, endemic transmission of C. diphtheriae was documented in some Native communities in Canada. Strains from the United States and Canada were found to be closely related to strains that circulated in the same areas during the 1970s and 1980s, suggesting ongoing endemic circulation in these populations. ^,

Epidemiology in Europe and the Rest of the World

Similar to the United States, Europe experienced a decline in the number of diphtherias cases with the implementation of widespread vaccination in the 1930s to 1950s. Despite these efforts, a major epidemic began and subsequently spread throughout the Newly Independent States (NIS) of the former Soviet Union, with more than 157,000 cases and 5000 deaths reported between 1990 and 1998. ^, The cause of the epidemic was likely multifactorial; contributing factors including a decline in childhood vaccination acceptance and coverage, exaggerated physicians fears about vaccine adverse events, the use of three doses of Td (diphtheria and tetanus toxoids—adult) instead of DTP (diphtheria, tetanus, pertussis-child) in the childhood immunization schedule, and the absence of an adult Td immunization program. ^, Delayed recognition of cases, belated public health response, and changing social conditions also helped to facilitate rapid spread of disease. The epidemic peaked between 1994 and 1995 and subsequently was brought under control by increasing immunization coverage with diphtheria toxoid among both children and adults. ^,

Since the conclusion of the NIS epidemic, cases reported in European countries are typically sporadic, although there are areas of continued endemic transmission such as Latvia. From 2014 to 2018, 38–65 diphtheria cases were reported annually among the 30 countries who report to the European Centre for Disease Prevention and Control. Approximately a quarter of the 63 cases reported in 2018 were considered imported from endemic regions, and interestingly, more than half were caused by toxigenic C. ulcerans .

With regard to low- and middle-income countries, a steady decrease in the incidence of diphtheria occurred after the implementation of routine vaccination programs, supported by the establishment of the World Health Organization (WHO) Expanded Programme on Immunization (EPI) in 1974. Globally, diphtheria cases reported to WHO declined by >85% from 1980 to 2000, from 97,164 to 11,625 annual cases, reaching a nadir of 4333 cases in 2006 ( Fig. 21.2 ). The annual number of reported cases generally remained at this level until 2014, when 7774 cases were reported; in the years since, the number of cases reported has been increasing, with 22,986 cases reported in 2019. While the number of cases reported in 2020 declined to 10,111, this may not represent an accurate count: the COVID-19 pandemic may have resulted in reduced patient presentation to healthcare for non-COVID-19 causes, or it may have resulted in decreased detection and reporting of non-COVID-19 diseases by public health entities due to prioritization of COVID-19 response over other activities.

Contributing to the recent diphtheria resurgence are a number of outbreaks, centered in countries with persistently low vaccination coverage, or civil unrest associated with a breakdown in public health infrastructure. Between 2010 and present, large laboratory-confirmed outbreaks have occurred in Haiti, Venezuela, Indonesia, Laos, Thailand, South Africa, and Yemen. ^{, ,} Most recently during 2017–2019, a significant outbreak occurred among Forcibly Displaced Myanmar National persons, in camps located in Cox’s Bazaar, Bangladesh. ^, The recurrence of outbreaks highlights the need to maintain or increase protective immunity through diphtheria toxoid vaccination.

With regard to geography, countries of the WHO South-East Asia Region have contributed the highest proportion (40–99%) of reported cases in comparison with other WHO regions since 2000. In particular, cases reported from India have represented 42–84% of the global burden annually from 2000 to 2019.

Age-related epidemiologic trends of diphtheria have changed with the introduction of diphtheria toxoid immunization programs. Prior to vaccine introduction, diphtheria was predominantly reported in children, primarily less than 15 years of age. With implementation of routine vaccination, the burden of disease has shifted to older children and adults, likely reflecting waning immunity of vaccination and a decline in environmental exposures that would have resulted in boosting. ^,

Even though the burden of diphtheria appears to be increasing, these case numbers may actually be an underestimate, due to limitations in surveillance. Globally, a small proportion of countries do not have any surveillance for diphtheria, while the majority only collect aggregate data (total number of cases). Additionally, case numbers are not uniformly reported to WHO each year by all countries; a recent analysis examining case data reported to WHO from 2000 to 2017 found that data were missing in nearly 20% of possible reporting years across all countries, with the highest percentage of missing (40%) from the WHO Africa Region. Implementation of standard surveillance, particularly in low- and middle-income countries, is critical to better understanding the burden of diphtheria. To that end, WHO recently shared recommendations for diphtheria surveillance, including guidance for national case-based surveillance.

High Risk Groups

Unvaccinated or inadequately vaccinated persons are at risk for diphtheria, regardless of age. Unvaccinated or undervaccinated preschool and school-age children are most often affected by respiratory diphtheria, while it is rare in infants younger than 6 months of age, presumably because of the presence of maternal antibody.

Although no differences in diphtheria incidence were noted by sex in the prevaccine era, an increased risk of diphtheria among women was reported in several outbreaks among adults in the 1940s ^, and subsequently an increased risk of diphtheria among women was observed in Russia and some of the other countries of the former Soviet Union during the NIS epidemic in the 1990s. ^, Subsequent examination of data regarding susceptibility to diphtheria by sex has been conflicting, with some suggesting that gender-specific immune responses to vaccination may play a role, and others finding that there was no difference between sex in measured antibody titers to diphtheria toxin.

Reservoirs of Disease

Historically, humans were presumed to be the sole reservoir of C. diphtheriae , however the bacteria has been recovered from other animals, including infected horses and dogs, without confirmed transmission from humans.