Acute Phase Pulmonary Responses to a Single Intratracheal Spray Instillation of Magnetite (Fe3O4) Nanoparticles in Fischer 344 Rats

Iron nanomaterials are of considerable interest for application to nanotechnology-related fields including environmental catalysis, biomedical imaging, drug delivery and hyperthermia, because of their superparamagnetic characteristics and high catalytic abilities. However, information about potential risks of iron nanomaterials is limited. The present study assessed pulmonary responses to a single intratracheal spray instillation of triiron tetraoxide nanoparticles (magnetite) in rats. Ten-week-old male and female Fischer 344 rats (n=5/group) were exposed to a single intratracheal spray instillation of 0 (vehicle), 5.0, 15.0 or 45.0 mg/kg body weight (BW) of magnetite. After 14 days, the rats were sacrificed, and biological consequences were investigated. The lung weights of the 15.0 and 45.0 mg/kg BW male and female groups were significantly higher than those of the control groups. The lungs of treated rats showed enlargement and black patches originating from the color of magnetite. The typical histopathological changes in the lungs of the treated rats included infiltration of macrophages phagocytosing magnetite, inflammatory cell infiltration, granuloma formation and an increase of goblet cells in the bronchial epithelium. The results clearly show that instilled magnetite causes foreign body inflammatory and granulating lesions in the lung. These pulmonary responses occur in a dose-dependent manner in association with the increase in lung weight.     

Serum concentration of surfactant protein D in horses with lower airway inflammation

Reasons for performing study: Surfactant protein D (SP‐D), mainly synthesised by alveolar type II cells and nonciliated bronchiolar cells, is one important component of innate pulmonary immunity. In man, circulating concentrations of SP‐D are routinely used as biomarkers for pulmonary injury. To date, serum SP‐D levels have only been investigated in horses in an experimental model of bacterial airway infection. Objectives: To compare serum SP‐D concentrations at rest and after exercise in horses with and without inflammatory airway disease (IAD). Methods: Venous blood samples were collected from 42 Standardbred racehorses at rest and 60 min after performing a standardised treadmill exercise test. Tracheal wash and bronchoalveolar lavage fluid (BALF) samples were collected after exercise. Based on BALF cytology, 22 horses were defined as IAD‐affected and 20 classified as controls. Serum SP‐D concentrations were assessed using a commercially available ELISA kit and statistically compared between groups of horses and sampling times. Results: Serum concentrations of SP‐D in IAD‐affected horses were significantly higher than those of control horses, both at rest and after exercise. Within the IAD‐affected group, no significant correlation was found between serum SP‐D concentrations and BALF cytology. Within each group of horses (IAD and control), no significant influence of exercise was found on serum SP‐D levels. Conclusions: This is the first study determining serum SP‐D concentrations in a noninfectious, naturally occurring form of lower airway inflammation in horses. The results highlight that IAD is associated with a detectable, though moderate, increase of circulating SP‐D levels. Potential relevance: Serum concentration of surfactant protein D could represent a potentially valuable and readily accessible blood biomarker of equine lower airway inflammation.     

Two-week Toxicity of Multi-walled Carbon Nanotubes by Whole-body Inhalation Exposure in Rats

To evaluate pulmonary toxicity of multi-walled carbon nanotubes (MWCNTs), F344 rats of both sexes were exposed by inhalation to 0.2, 1 or 5 mg/m³ MWCNT aerosol for 6 h/day, 5 days/week for 2 weeks using a whole-body exposure system. At the end of the 2-week exposure period, one-half of the rats were necropsied, and at the end of an additional 4-week postexposure period, the remaining rats were necropsied. MWCNTs were deposited in the lungs of all MWCNT-exposed groups and mostly remained in the lungs throughout the 4-week postexposure period. Granulomatous changes in the lung were found in the rats exposed to 5 mg/m³ MWCNTs, and these changes were slightly aggravated at the end of the 4-week postexposure period. In the bronchoalveolar lavage fluid (BALF), the numbers of neutrophils, percentages of bi- and multinucleated alveolar macrophages, levels of ALP activity and concentrations of total protein and albumin were elevated in the rats exposed to 1 and 5 mg/m³ MWCNTs. At the end of the 4-week postexposure period, the values of the BALF parameters tended to remain elevated. In addition, goblet cell hyperplasias in the nasal cavity and nasopharynx were observed in the rats exposed to 1 and 5 mg/m³ MWCNTs, but these lesions had largely regressed by the end of the postexposure period. Based on the histopathological and inflammatory changes, the no-observed-adverse-effect level (NOAEL) for inhalation of MWCNTs for 2 weeks was 0.2 mg/m³.     

Correlation of clinical score, intrapleural pressure, cytologic findings of bronchoalveolar fluid, and histopathologic lesions of pulmonary tissue in horses with summer pasture-associated obstructive pulmonary disease

OBJECTIVE: To correlate clinical score, intrapleural pressure, cytologic findings of bronchoalveolar lavage fluid (BALF), and histologic lesions of pulmonary tissue in horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD). ANIMALS: 8 adult horses affected with SPAOPD and 6 adult horses without evidence of respiratory tract disease. PROCEDURE: Clinical score, change in intrapleural pressure (deltaPpl) during tidal breathing, results of cytologic examination and bacteriologic culture of BALF, and results of histologic examination of pulmonary parenchyma were evaluated. RESULTS: Clinical scores for SPAOPD-affected horses (median, 5.75; range, 4.0 to 7.5) were significantly greater, compared with clinically normal horses (median, 2.0; range, 2.0 to 3.0). Cytologic examination of BALF from SPAOPD-affected horses revealed predominantly nondegenerate neutrophils. Histologic lesions were identified throughout pulmonary tissue and included severe accumulation of mucus and neutrophils within the small airways, metaplasia of bronchiolar goblet cells, and mild peribronchial infiltrate. Histologic examination of specimens collected via percutaneous biopsy was predictive of disease and corresponded to findings at postmortem examination. Clinical score and deltaPpl were highly correlated with mucus accumulation in the airways of affected horses. Peribronchial inflammatory infiltrate correlated with percentage of neutrophils in BALF of affected horses. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical scoring and deltaPpl provided valid estimates of disease severity. Findings from cytologic examination of BALF of SPAOPD-affected horses varied, although, in most instances, it was diagnostically useful. Severe mucus accumulation in the airways was the most remarkable histopathologic finding in SPAOPD-affected horses. Examination of biopsy specimens collected from pulmonary parenchyma was consistently useful in diagnosing SPAOPD.     

Effect of repetitive bronchoalveolar lavage on cytologic findings in healthy dogs

OBJECTIVE: To determine reference values for cytologic examination results of bronchoalveolar lavage fluid (BALF) and to investigate effects of repeated lavages on pulmonary health and on results of cytologic examination of BALF in dogs. ANIMALS: 16 healthy adult Beagles. PROCEDURE: All dogs underwent pulmonary lavage to obtain BALF. Eleven dogs were repeatedly lavaged 6 times at 5- to 7-week intervals. Analyses for total and differential cell counts and for viability of cells before and after cell processing were performed. Arterial blood gas analysis before and after bronchoalveolar lavage was used to study the safety of the lavage procedure. Histologic and radiologic examinations were used to study effects of repeated lavages on pulmonary health. RESULTS: Mean (+/- SD) cell count was 104 +/- 69 cells/microl, comprising 75 +/- 7% alveolar macrophages, 13 +/- 6% lymphocytes, 5 +/- 4% neutrophils, 4 +/- 5% eosinophils, 2 +/- 2% mast cells, 0.6 +/- 0.7% epithelial cells, and 0.3 +/- 0.4% plasma cells. Centrifugation of samples and washing of cells caused significant cell loss (59 +/- 13%). Repeated lavages did not cause significant variations in cell counts of BALF or results of arterial blood gas analysis, thoracic radiography, or histologic examination of pulmonary specimens. Only a moderate, although significant, decrease in arterial oxygen content was observed after bronchoalveolar lavage. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis indicated that several lavages performed at 5- to 7-week intervals can safely and reliably be used to study the kinetics of pathologic processes in pulmonary tissues or for evaluation of therapeutic efficacy.     

Comparison of acute inhalation toxicity of sulfuric acid by the inhalation and intratracheal instillation methods

Recently, intratracheal instillation has been focused on as a simple, low-cost alternative to the inhalation method. In this study, intratracheal instillation of sulfuric acid, a typical acidic compound, was performed to compare the acute toxicity of acidic compounds that could cause damage to the respiratory system between intratracheal instillation and inhalation. Sulfuric acid was administered to male rats at doses of 0.7, 2, 7, 20, and 60 mg/kg by dividing the total dose into four doses. General condition and body weight were examined up to 14 days after administration, and macropathological and histopathological examinations were performed. The half-lethal dose was then estimated. All animals administered 20 and 60 mg/kg sulfuric acid and one animal administered 2 mg/kg sulfuric acid died within 4 h after administration. No abnormalities were observed in other animals. At 20 and 60 mg/kg, multiple red foci or diffuse red areas were macroscopically observed in the lungs. In these lesions, histopathologically, clefts between the mucosal epithelium and basement membrane and necrosis of the alveolar epithelium were observed. Deaths in these groups may have resulted from lung injury. No notable changes were observed in other animals. Therefore, the half-lethal dose of sulfuric acid by intratracheal instillation was estimated as 7–20 mg/kg. The acute toxicity by intratracheal instillation was evaluated with two-fold sensitivity since the exposure at the half-lethal sulfuric acid concentration in inhalation studies was calculated as 43.2 mg/kg.     

An experimentally induced Chlamydia suis infection in pigs results in severe lung function disorders and pulmonary inflammation

This study was aimed at evaluating the pathophysiology of pulmonary dysfunctions and inflammatory consequences of an acute respiratory chlamydial infection induced experimentally in conventionally raised pigs (aged 39-44 days). Eight animals were exposed to Chlamydia suis (C. suis) and four non-infected animals served as controls. The total observation period was from seven days before challenge to seven days post exposure. While non-infected control pigs did not exhibit any clinical symptoms, animals exposed to C. suis developed fever and were severely respiratory distressed within the first week after exposure. After C. suis infection, pulmonary dysfunctions were characterised by a significant decrease in the diffusion capacity of the lung (i.e. transfer factor of the lung for carbon monoxide; TL CO), a significant increase in the functional residual capacity (FRC), and significant changes in the pattern of ventilation (respiratory rate increased while the tidal volume decreased). In exhaled breath condensate (EBC), leukotriene B(4) (LTB(4)) and interleukin 6 (IL-6) showed a tendency to increase after infection. In the broncho-alveolar lavage fluid (BALF) of C. suis infected pigs, the activity of matrix metalloprotease 9 (MMP-9) was found to be increased compared to controls. BALF cytology was characterised by increased numbers of granulocytes and activated lymphocytes. Pulmonary inflammation in infected pigs was confirmed by post mortem histology. A prominent dissemination of chlamydial bodies in the lung was accompanied by an influx of macrophages, granulocytes and activated T-cells. Data obtained in this study provide new insight into the pathogenesis of acute respiratory chlamydial infections in pigs.     

Comparison of Biological Responses in Rats Under Various Cigarette Smoke Exposure Conditions

A variety of exposure regimens of cigarette smoke have been used in animal models of lung diseases. In this study, we compared biological responses of smoke exposure in rats, using different smoke concentrations (wet total particulate matter [WTPM]), daily exposure durations, and total days of exposure. As a range-finding acute study, we first compared pulmonary responses between SD and F344 strains after a single nose-only exposure to mainstream cigarette smoke or LPS. Secondly, F344 rats were exposed to cigarette smoke for 2 or 13 weeks under the comparable daily exposure dose (WTPM concentration x daily exposure duration; according to Haber’s rule) but at a different WTPM concentration or daily exposure duration. Blood carboxylhemoglobin was increased linearly to the WTPM concentration, while urinary nicotine plus cotinine value was higher for the longer daily exposure than the corresponding shorter exposure groups. Gamma glutamyl transferase activity in bronchoalveolar lavage fluid (BALF) was increased dose dependently after 2 and 13 weeks of cigarette smoke exposure, while the neutrophil content in BALF was not increased notably. Smoke-exposed groups showed reduced body weight gain and increased relative lung and heart weights. While BALF parameters and the relative lung weights suggest pulmonary responses, histopathological examination showed epithelial lesions mainly in the upper respiratory organs (nose and larynx). Collectively, the results indicate that, under the employed study design, the equivalent daily exposure dose (exposure concentration x duration) induces equivalent pulmonary responses in rats.     

Pulmonary inflammation due to exercise-induced pulmonary haemorrhage in Thoroughbred colts during race training

This study investigated the putative roles of inflammation and platelet-activating factor (PAF) in exercise-induced pulmonary haemorrhage (EIPH). Two-year-old Thoroughbred colts (n=37) were exercised on a racetrack for 5months before commencement of the study. Each colt was then exercised at 15-16m/s over 800-1000m and broncho-alveolar lavage fluid (BALF) was collected 24h later. The colts were subsequently divided into two groups on the basis of BALF analysis; an EIPH-positive group (presence of haemosiderophages, n=23) and an EIPH-negative group (absence of haemosiderophages, n=14). BALF from the EIPH-positive group had a significantly higher protein concentration (0.39±0.28 vs. 0.19±0.12mg/mL, P=0.031), higher PAF bioactivity (0.18±0.12 vs. 0.043±0.05 340:380nm ratio, P=0.042) and a higher lipid hydroperoxide concentration compared to the EIPH-negative group. There was also a lower nitrite concentration and reduced production of superoxide anion and hydrogen peroxide by alveolar macrophages in the EIPH-positive group. There was evidence of pulmonary inflammation and a decreased innate immune response of alveolar macrophages in EIPH-positive colts compared with the EIPH-negative group.     

Arachidonate metabolites in bronchoalveolar lavage fluid from horses with and without COPD.

Arachidonate metabolites were measured in bronchoalveolar lavage fluid (BALF) from horses with (N = 4) and without (N = 7) chronic obstructive pulmonary disease (COPD). Prostaglandin (PG) D2, leukotriene (LT) B4 and LTC4 were present in highest concentrations in BALF from clinically normal horses. Concentrations of PGE2 and PGF were significantly higher in BALF from horses with COPD than in BALF from normal horses, but no differences were detected in thromboxane B2, 6-keto-PGF1 alpha, PGD2, LTB4 or LTC4.     

Toxicity of Nicotine by Repeated Intratracheal Instillation to F344 Rats

In vivo, nicotine in cigarette smoke induces various effects not only on the respiratory system but also the central and peripheral nerve systems, circulatory organs and digestive organs, and there is a possibility of promotion of lung tumorigenesis. The present experiment was conducted to examine histopathological changes caused by nicotine in the lung with repeated intratracheal instillation (i.t.). Six-week-old male F344 rats were administered nicotine by i.t. at doses of 0.05, 0.1 and 0.2 mg nicotine/rat every 3 weeks beginning at week 4, for up to a total of 9 times and were then sacrificed at week 30. The total number of administrations, total dose of nicotine and effective number of rats were 9 times, 0.45 mg and 5 rats and 4 times, 0.20 mg and 5 rats for the 0.05 mg nicotine/rat group; 3 times, 0.30 mg and 5 rats and 4 times, 0.40 mg and 3 rats for the 0.1 mg group; and 3 times, 0.60 mg and 3 rats for the 0.2 mg group, respectively. As a control group, 5 rats were administered 0.2 ml saline/rat 9 times. Some rats administered 0.1 and 0.2 mg nicotine suffered convulsions just after administration. Histopathologically, though proliferative changes were not observed, neutrophil infiltration, edema and fibrosis in the lung were induced by nicotine. In conclusion, repeated treatment of nicotine promoted neurologic symptoms in the acute phase, and strong inflammation in the lungs in the chronic phase, even at a low dose. Toxicity of nicotine is suggested to depend not on total dose of nicotine in the experiment but rather on repeated injury with consecutive administration.     

Elevated levels of fragmented laminin-5 gamma2-chain in bronchoalveolar lavage fluid from dogs with pulmonary eosinophilia

Inflammation causes epithelial cell sloughing and basement membrane (BM) exposure in canine pulmonary eosinophilia (PE), leading to degradation of the epithelial cell attachment component, laminin-5 gamma2-chain, into small molecular weight fragments. The subsidence of inflammation after treatment down-regulates degradation. Laminin-5 gamma2-chain levels and molecular forms in bronchoalveolar lavage fluid (BALF) were analysed semiquantitatively by Western immunoblotting to compare PE affected (n=20) and healthy dogs (n=16) as well as PE dogs (n=6) before and after corticosteroid treatment. PE dogs expressed significantly elevated levels of total (P<0.01), 36 kDa (P<0.05) and 53 kDa (P<0.05) laminin-5 gamma2-fragments. The 36 Da fragment decreased significantly (P<0.05) after treatment. The laminin-5 gamma2-chain degradation products may be linked to epithelial cell sloughing and BM exposure or healing.     

Leukocyte counts in bronchoalveolar lavage fluid obtained from normal and Maedi–Visna‐infected sheep

Background: Bronchoalveolar lavage has proven helpful for the diagnosis of certain ovine diseases of the lungs. There is insufficient data concerning the leukocyte profile of bronchoalveolar lavage fluid (BALF) from MaediVisna infected sheep. Objective: The objective of this study was to assess the differential leukocyte profile of BALF associated with Maedi virus infection in sheep and to determine whether cytologic examination of BALF is an effective way to diagnose Maedi or determine the severity of lung lesions. Methods: BALF and serum samples were analyzed from 400 sheep. Sediment smears of bronchoalveolar lavage were stained with Diff‐Quik and examined microscopically to obtain a 200‐cell differential cell count. Serum was tested using a commercial kit for Maedi–Visna virus antibodies. Lung samples obtained at the time of slaughter were weighed and examined histologically. Results: Maedi‐infected sheep (n=267; seropositive with lung lesions) had a significantly higher percentage of lymphocytes and lower percentage of macrophages in BALF than normal sheep (n=133; seronegative and no lung lesions). These differences were significantly more severe in animals with advanced vs moderate lung lesions. Using classification trees, a cut‐off of 13.5% lymphocytes was predictive of Maedi infection and a cut‐off of 24.5% lymphocytes was predictive of advanced lung lesions. Conclusions: Cytologic examination of BALF is useful for the clinical diagnosis of Maedi in sheep and provides important information about the severity of the lung lesions.     

Detection of respiratory pathogens in porcine lung tissue and lavage fluid

The objective of this study was to compare the detection rate of bacterial agents in bronchoalveolar lavage fluid (BALF), taken without visual control, to that in affected lung tissue obtained from the same pig at necropsy. BALF and affected lung tissue were examined for Mycoplasma hyopneumoniae using PCR, and standard cultural methods were used for Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida and Streptococcus suis. All pigs with a history of respiratory symptoms were submitted as live animals for routine diagnostic examination. In each animal the site of lavage, marked by injecting methylene blue, differed from the site of pneumonic lesions. M. hyopneumoniae was detected more frequently in lung tissue than in BALF in cases with moderate or severe lung lesions. The detection rates of M. hyopneumoniae were higher in the BALF of pigs with mild lesions. Cultural examination of BALF was at least as satisfactory as affected lung tissue for detecting B. bronchiseptica, H. parasuis and P. multocida.     

Pulmonary dysfunction in neonatal calves after intratracheal inoculation of small volumes of fluid

Intratracheal instillation of 20 ml of room temperature (21 to 24 C) fluid in anesthetized neonatal calves resulted in rapid onset of reversible pulmonary dysfunction. Arterial O2 tension and dynamic compliance decreased, whereas pulmonary arterial pressure, pulmonary vascular resistance, alveolar arterial O2 difference, and total pulmonary resistance increased from base-line values. Abnormalities of gas exchange and pulmonary mechanics were induced by intratracheal fluid instillation whether or not Pasteurella haemolytica was in the inoculum. Physical manipulation of the calf without intratracheal fluid instillation (sham inoculation) did not influence pulmonary function. Bilateral vagotomy eliminated the increase in pulmonary resistance and the decrease in dynamic compliance, but did not eliminate hypoxemia, increased alveolar arterial O2 difference, or pulmonary hypertension recorded after intratracheal fluid instillation. Seemingly, changes in pulmonary mechanics are mediated via the vagus nerve. However, one or more additional mechanisms must be responsible for the hypoxemia and pulmonary hypertension.     

Collagenolytic activity in bronchoalveolar lavage fluid in canine pulmonary eosinophilia

We studied and characterized the collagenolytic matrix metalloproteinases (MMP-8 and MMP-13) in the pathogenesis of canine pulmonary eosinophilia (PE). Twenty dogs with PE and 16 healthy control dogs underwent similar clinical examination and collection of bronchoalveolar lavage fluid (BALF). Analyses of total cell and differential cell counts and collagen I degradation with and without aminophenyl mercuric acetate (APMA) treatment were performed. Correlations between cell counts and percentage of degraded collagen I in BALF were studied. Collagenase activity detected in BALF was characterized by Western immunoblotting for collagenase-2 (MMP-8) and collagenase-3 (MMP-13), and their cellular location was studied by immunocytochemical means. Collagenolytic activity was significantly increased in cell-free and native BALF of PE dogs compared to healthy controls. APMA treatment had no significant effect on BALF collagenase activity, indicating that collagenolytic activity occurred in diseased BALF in vivo in active form. Western immunoblotting identified the presence of MMP-8 and MMP-13 immunoreactivities, of which the latter was converted to active form. Major immunoreactivity for MMP-8 was observed in macrophages and epithelial cells, and major immunoreactivity for MMP-13 was observed in macrophages. A significant positive correlation was noted between the percentage of degraded collagen I and the counts of eosinophils, macrophages, lymphocytes, and mast cells. These findings suggest that the up-regulation of collagenolysis eventually contributes to pulmonary tissue destruction in canine PE.     

Characterisation of the acute and reversible airway inflammation induced by cadmium chloride inhalation in healthy dogs and evaluation of the effects of salbutamol and prednisolone

The aims of this study were firstly to characterise a model of subclinical and reversible bronchial inflammation induced by cadmium chloride inhalation in healthy dogs and then to examine the effect of prednisolone or salbutamol treatment on the resulting bronchitis. The model characterisation and the effects of treatment were studied using clinical symptoms, haematology, thoracic radiography, bronchoscopy and bronchoalveolar lavage, barometric whole-body plethysmography and histamine broncho-provocation tests. In addition, the activity of matrix metalloproteinases (MMP)-2 and -9 were determined in serum and bronchoalveolar lavage fluid (BALF). Cadmium inhalation induced: (1) a transient bronchial inflammation, dominated by neutrophils; (2) a neutrophilia of the blood that persisted for up to 4 weeks; (3) a transient increased bronchial reactivity, and (4) a significant increase in MMP-9 activity in the BALF. Prednisolone treatment reduced the influx of inflammatory cells into the BALF, but not significantly, had no effect on pulmonary function, and did not reduce of airway hypersensitivity. Salbutamol had almost no effect on any of the parameters investigated.     

Antioxidant and inflammatory responses of healthy horses and horses affected by recurrent airway obstruction to inhaled ozone

REASONS FOR PERFORMING STUDY: Inhaled ozone can induce oxidative injury and airway inflammation. Horses affected by recurrent airway obstruction (RAO) have a decreased pulmonary antioxidant capacity, which may render them more susceptible to oxidative challenge. It is currently unknown whether RAO-affected horses are more susceptible to oxidative stress than those unaffected by RAO. OBJECTIVES: To determine whether ozone exposure induces greater oxidative stress and airway inflammation in RAO-affected horses in remission than in healthy horses. METHODS: Seven healthy control horses and 7 RAO-affected horses were exposed to 0.8 ppm ozone for 2 h at rest. RESULTS: At baseline, bronchoalveolar lavage fluid (BALF) ascorbic acid concentrations were lower in RAO-affected horses than healthy controls. Ozone appeared to preferentially oxidise glutathione rather than ascorbic acid 6 h after exposure. Individual healthy and RAO-affected horses demonstrated oxidation of BALF glutathione after ozone exposure. Overall, RAO-affected horses did not demonstrate increased oxidative stress following ozone exposure, compared with healthy horses. Ozone did not induce significant airway inflammation in either group. CONCLUSIONS: RAO-affected horses in remission are not more sensitive to ozone despite a decreased pulmonary antioxidant capacity. Sensitivity to ozone appears to be independent of initial pulmonary antioxidant status. POTENTIAL RELEVANCE: Horses with high susceptibility to oxidative stress may benefit from antioxidant supplementation.     

Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with “exceptional” gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these “exceptional” lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.