Melioidosis has a diverse clinical presentation in humans. This may be affected by individual characteristics of the infecting strain, dose, and route of infection, which is rarely known in naturally occurring cases. These unknowns make it very difficult to study the pathogenesis of melioidosis despite the large number of individual case reports and several retrospective and prospective studies. Basic pathogenesis research has been largely restricted to murine models, but additional models are necessary for a more complete understanding of the pathogenesis of infection with B. pseudomallei, which has a very broad host range. Additionally, new animal model systems are also important for the development and testing of novel therapeutics and vaccines.
Patients with melioidosis typically present with protean signs. Since there is no “typical” presentation, the subdivision of cases is highly variable in the human medical literature, with some papers grouping patients by duration of signs (acute vs. chronic), the presence or absence of septic shock/bacteremia, or the primary organ system involved. Pneumonia, with (76%) or without (45%) septic shock, accounts for the largest percentage of clinical presentations of naturally occurring human melioidosis 
. In pulmonary cases, signs that are typically present include fever (50–100% of patients) 
and leukocytosis (70% of patients) 
In our goat model, the only consistent clinical sign of infection was fever. The degree of pyrexia appeared to parallel the increase in granulocyte count over the first seven to nine days, with peaks on day 1 and around day 7. After day 9, temperatures tended to decrease, but this decrease was not necessarily paralleled in the granulocyte count (see and ). The consistency of these alterations is expected early in infection when the dominant variables of route of infection, dose, and strain are all controlled for by the nature of experimental infection. As disease progresses over time, individual host factors may become more important and account for the greater variability seen.
Other clinical signs included cough and oculonasal discharge. Nasal swabs, including those from goats with nasal discharge, were typically culture negative for B. pseudomallei
except in the case of goat 16, where increasingly heavy discharge and growth of B. pseudomallei
was associated with the progression towards terminal disease. Light positive growth seen on day 1 was interpreted as residual bacteria from intratracheal infection rather than dissemination or expectorated pus. This low rate of nasal shedding suggests that horizontal spread among animals via nasal secretions would be unlikely or insignificant. This appears similar to human disease where there are very few reports of person-to-person transmission 
even though 68% of patients with evidence of pulmonary involvement are positive on sputum culture 
Hematogenous dissemination, the only mechanism consistent with the observed pattern of multiorgan involvement, was not directly confirmed, likely because of the transient nature of bacteremia. Positive blood cultures were only seen in goat 16, which was associated with the progression to terminal disease. The bacteremia observed in goats appears similar to the bacteremia seen in human patients, in which only 50% of patients are culture positive, and of the culture positive patients the median bacteremia is 1.1 CFU/ml 
. Urine collected at postmortem examination was only positive in one of the four goats with renal abscessation. The goat with a positive urine culture had terminal disease at the time of collection, which could suggest that urinary shedding is a late event and/or indicative of a more severe or advanced expansion of renal abscesses into the pelvis. This could be consistent with the finding that the presence of a positive urine culture is a negative prognostic indicator in human melioidosis patients 
. Counts ranging from 104
CFU/ml were also seen in a small number of goats used in the dose determination portion of this study (data not shown), which is comparable to counts seen in human patients 
Thoracic radiographic findings in human melioidosis are highly variable and the incidence of specific findings also varies considerably among retrospective imaging studies of melioidosis. Radiographic signs in acutely presenting cases often have local, patchy, alveolar infiltrates, or disseminated nodular lesions, which are suggestive of metastatic (hematogenous) spread 
. Acute cases tend to have much more rapid deterioration and death 
. The findings in subacute to chronic presentations are more variable, with some studies reporting the absence of a predominant lesion 
, while others report findings very similar to what is seen in acute cases 
. The upper lobes appear to be more affected in melioidosis, especially in subacute/chronic cases 
. The predominant radiographic changes seen in the goats after aerosol exposure were bronchointerstitial infiltrates indicative of airway inflammation; pulmonary nodules were often initially identified in the caudal lungs and subsequently spreading to all lung lobes. This distribution was as expected with aerosol delivery (with or without secondary septicemic seeding of the lungs), but radiographically could have the same appearance as abscesses formed from the hematogenous spread of bacteria. Therefore, it is possible that the nodular appearance in human cases could be the result of inhalational and/or septicemic disease. Despite aerosol delivery of bacteria and acute fever, there was not rapid progression of disease radiographically or clinically. This is partially a function of dose as was seen in dose determination studies, where a delivered dose of 107
CFU produced fulminant pulmonary disease, necessitating euthanasia 48 h PI. Additionally, the use of healthy animals, without any predisposing risk factors for melioidosis, could have limited the development of rapidly progressive disease.
Organ involvement in melioidosis can be highly variable in both humans and goats. In humans, the lungs are the most commonly affected organ, but virtually any organ can be affected 
. Data concerning organ distribution of naturally occurring melioidosis lesions seen in goats has been described in abattoir studies from Malaysia 
and Australia 
. The pattern of organ involvement seen in the goats in these studies 
, previous experimental infections 
, and our current model is generally similar to what is seen in human disease, with the spleen, lung, kidney, and liver being commonly affected.
One notable difference in organ involvement and lesion severity observed in goats in this study compared to mice and humans was the rarity of gross hepatic abscesses and only mild severity of microscopic changes within the liver. Previous reports of both natural and experimental melioidosis in goats have had variable findings in regards to liver lesions, with some reporting no lesions 
and others finding hepatic lesions to be quite common 
. This finding may be a result of strain variation, individual host/population factors, or the duration of infection. Hepatic (and splenic) abscesses are much more common in Thai patients than Australian patients 
, which again may relate to strain variation and duration of illness.
In the current study, the lungs were the primary site of infection. Dissemination to extrapulmonary tissues appeared to be predominantly a function of time, with greater numbers of organs involved at the later time points. Dissemination to sites other than lymph nodes draining the pulmonary system was not grossly evident before day 14 with the exception of one small adrenal abscess seen in one goat on day 7. The histologic evidence suggests that the timing of dissemination was a result of the size and extent of the pulmonary pyogranuloma formation. After aerosol delivery, mucopurulent bronchopneumonia rapidly developed and soon progressed to a more severe fibrinopurulent to bronchointerstitial pneumonia with pyogranuloma formation evident radiographically, grossly, and histologically by day 7. Within the lungs, the lesions spread along interlobular septa and subpleural stroma inducing a local leukocytoclastic vasculitis where the pyogranulomas encroached upon and eventually invaded the vessel (). The vasculitis appeared to peak in severity around day 7. The rise in temperatures around day 7 as well as the gross appearance of the majority of the extrapulmonary lesions on day 14 or later supports the central role of the vasculitis in hematogenous dissemination of B. pseudomallei.
Vasculitis also appears to be the central pathologic step in the establishment and progression of pyogranuloma development in extrapulmonary organs. Vasculitis within an adrenal vessel was noted in association with abscess formation and invasion of the parenchyma. Splenic lesions in the capsule and parenchyma were also seen in association with vasculitis. The predominance of capsular pyogranulomas appears to correlate with the capsulitis observed histologically. The renal lesions suggest that the point of entry for B. pseudomallei is the glomerulus, with the earliest detectable lesion being hypercellular glomeruli with neutrophil infiltration. The lesions then suppurate and extend into tubules creating tubulointerstitial disease and ultimately result in the formation of pyogranulomas. The testicle was the only organ with lesions that had evidence of hemorrhage in association with the vasculitis. The significance of this finding is unclear at this time since a testicular lesion was only seen in one goat, but lends support to vasculitis being a central event in pathogenesis.
To the authors’ knowledge, vasculitis has not previously been reported as a feature of human disease or in murine models. It has been reported in goats in association with central nervous system lesions 
. There are a limited number of papers with detailed histopathologic descriptions of human disease 
, none of which report vasculitis. The majority of these reports are based on acute fatal cases with lesions that are more suppurative in nature. Zones of hemorrhage, particularly surrounding pulmonary lesions are frequently reported in acute human cases 
, but have not been reported to be associated with vasculitis. A more recent paper, which also included a number of surgical biopsy samples from chronic cases in addition to acute fatal cases, specifically noted that vasculitis was not found, and only reported hemorrhage being variably present in biopsy samples 
. The lack of vascular lesions in human cases may be a species difference or could reflect the skewed distribution of histologic samples in human cases.
Goats are generally believed to have a natural tendency towards more chronic disease and granulomatous type lesions in melioidosis 
, though disease ranging from acutely fatal to apparently self-curing lesions has been observed 
. While there are certain notable differences in caprine and human melioidosis, the ability of goats to survive the acute stages of melioidosis, contain B. pseudomallei
to more chronic lesions, and even potentially eliminate the organism, makes them a particularly interesting animal model of melioidosis. We have shown that melioidosis can be readily induced in goats following aerosol exposure. As expected, the extent of organ involvement seen was more variable in goats than in murine models, but was comparable to human disease and is likely to be a feature of any model with a truly heterogeneous outbred population. However, it appeared that organ involvement may become more consistent at later time points with more chronic disease in our caprine model.
The larger body size of goats allows for human-relevant clinical monitoring as well as longer-term serial evaluation of disease progression and therapy. We believe the caprine model will be a useful animal model for further investigation of the molecular pathogenesis and host response in melioidosis, as well as evaluation of preventative and therapeutic interventions.