Histopathological and immunohistochemical examination of the brains of rabid
dogs in the Philippines
Animals and direct FATs (dFATs) Seventy suspected rabid domestic dogs were submitted to the Research Institute for Tropical Medicine (RITM) in the Philippines for routine postmortem rabies diagnosis, which was confirmed by using dFAT. The data from individual dogs were recorded. Small transverse sections (2–3 mm thick) of the Ammon’s horn and medulla were excised. Touch imprint slides prepared from the excised surface of the sections were placed on cold acetone overnight for fixation. After fixation, the slides were air-dried at room temperature (RT). Then, 45 μL of fluorescein isothiocyanate conjugate (FITC) anti-rabies monoclonal antibody (Fujirebio ® , Malvern, PA, USA) was added to the slides. The slides were incubated for 30 min at 37°C in a high-humidity chamber. The slides were dipped and rinsed twice in PBS, and then washed with distilled water. Small amounts of the mounting medium (20% glycerol-Tris buffered saline pH 9.0) were placed on the slides before covering them with coverslips for examination. The slides were examined under a fluorescence microscope (80i; Nikon, Tokyo, Japan). Histopathological examination Brain tissue from rabid dogs ( n =70) containing cerebrum, hippocampus, thalamus, and brainstem was fixed in 10% neutral buffered formalin for at least 72 hr at RT. All the specimens were embedded in paraffin. The FFPE tissues were transversely sectioned (2–3 µm), stained with HE, or labeled using the IHC method described below. All brain samples were selected based on minimal postmortem changes, as determined by gross and histological observations. The samples from one beagle dog, aged 10 months and vaccinated against rabies, were used as the negative controls. The cerebrum, hippocampus, thalamus, and brainstem were available for all dogs. Immunohistochemistry The polymer-based immunohistochemical method was used to detect rabies viral antigens and cell types. Primary antibodies were utilized: anti-rabbit phosphoprotein (anti-P) , a CD3 antibody (Agilent DAKO, Kyoto, Japan) for T lymphocytes; a CD20 antibody (Thermo Fisher Scientific, Fremont, CA, USA) for B lymphocytes; and an antibody against ionized calcium-binding adaptor molecule 1 (Iba1) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for microglial cells . Briefly, to activate the antigen in tissue sections, 0.25% trypsin was used for 30 min at RT for anti-P, and microwave treatment at 170 W for 10 min for CD3 and CD20. Subsequently, 0.3% or 3% hydrogen peroxidase in methanol was used to remove endogenous peroxidase. Tissue sections were treated with 10% normal goat serum to block non-specific reactions before being incubated in a humidity chamber overnight at 4°C with primary antibodies (1:1,000 for anti-P, 1:400 for CD20, 1:500 for Iba1, and predilute CD3). The Envision + System HRP labeled polymer rabbit antibody (Agilent Dako) was used for CD3, CD20, and Iba1 detection. The Histofine ® Simple Stain Max PO (Rabbit) (Nichirei Biosciences, Tokyo, Japan) was used for anti-P and detection. Antigen-antibody reactions were visualized using 3,3'-Diaminobenzidine (DAB; Nichirei Biosciences). The tissue sections were counterstained with hematoxylin and mounted. Histopathological (HE) and IHC analysis The presence or absence of Negri bodies was recorded. Perivascular cuffing and glial nodules were visually semi-quantitatively graded on scales of 0 to 3 and 0 to 2, respectively. The number of anti-P immunoreactive neuronal cells was visually semi-quantitatively scored on a scale of 0 to 3 as follows: no positive neurons as 0, 1–30% neurons as 1, 30–60% neurons as 2, and 60–100% neurons as 3. All brain regions were independently analyzed by three pathologists, with discrepancies resolved by consensus. Statistical analysis The differences in median HE and anti-P scores among different brain locations were analyzed using the Kruskal-Wallis H Test. Post-hoc multiple comparisons were performed using Dunn’s test with Bonferroni correction. All statistical analyses were performed using JMP Pro 16 software (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at P <0.05.   Seventy suspected rabid domestic dogs were submitted to the Research Institute for Tropical Medicine (RITM) in the Philippines for routine postmortem rabies diagnosis, which was confirmed by using dFAT. The data from individual dogs were recorded. Small transverse sections (2–3 mm thick) of the Ammon’s horn and medulla were excised. Touch imprint slides prepared from the excised surface of the sections were placed on cold acetone overnight for fixation. After fixation, the slides were air-dried at room temperature (RT). Then, 45 μL of fluorescein isothiocyanate conjugate (FITC) anti-rabies monoclonal antibody (Fujirebio ® , Malvern, PA, USA) was added to the slides. The slides were incubated for 30 min at 37°C in a high-humidity chamber. The slides were dipped and rinsed twice in PBS, and then washed with distilled water. Small amounts of the mounting medium (20% glycerol-Tris buffered saline pH 9.0) were placed on the slides before covering them with coverslips for examination. The slides were examined under a fluorescence microscope (80i; Nikon, Tokyo, Japan).   Brain tissue from rabid dogs ( n =70) containing cerebrum, hippocampus, thalamus, and brainstem was fixed in 10% neutral buffered formalin for at least 72 hr at RT. All the specimens were embedded in paraffin. The FFPE tissues were transversely sectioned (2–3 µm), stained with HE, or labeled using the IHC method described below. All brain samples were selected based on minimal postmortem changes, as determined by gross and histological observations. The samples from one beagle dog, aged 10 months and vaccinated against rabies, were used as the negative controls. The cerebrum, hippocampus, thalamus, and brainstem were available for all dogs.   The polymer-based immunohistochemical method was used to detect rabies viral antigens and cell types. Primary antibodies were utilized: anti-rabbit phosphoprotein (anti-P) , a CD3 antibody (Agilent DAKO, Kyoto, Japan) for T lymphocytes; a CD20 antibody (Thermo Fisher Scientific, Fremont, CA, USA) for B lymphocytes; and an antibody against ionized calcium-binding adaptor molecule 1 (Iba1) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for microglial cells . Briefly, to activate the antigen in tissue sections, 0.25% trypsin was used for 30 min at RT for anti-P, and microwave treatment at 170 W for 10 min for CD3 and CD20. Subsequently, 0.3% or 3% hydrogen peroxidase in methanol was used to remove endogenous peroxidase. Tissue sections were treated with 10% normal goat serum to block non-specific reactions before being incubated in a humidity chamber overnight at 4°C with primary antibodies (1:1,000 for anti-P, 1:400 for CD20, 1:500 for Iba1, and predilute CD3). The Envision + System HRP labeled polymer rabbit antibody (Agilent Dako) was used for CD3, CD20, and Iba1 detection. The Histofine ® Simple Stain Max PO (Rabbit) (Nichirei Biosciences, Tokyo, Japan) was used for anti-P and detection. Antigen-antibody reactions were visualized using 3,3'-Diaminobenzidine (DAB; Nichirei Biosciences). The tissue sections were counterstained with hematoxylin and mounted.   The presence or absence of Negri bodies was recorded. Perivascular cuffing and glial nodules were visually semi-quantitatively graded on scales of 0 to 3 and 0 to 2, respectively. The number of anti-P immunoreactive neuronal cells was visually semi-quantitatively scored on a scale of 0 to 3 as follows: no positive neurons as 0, 1–30% neurons as 1, 30–60% neurons as 2, and 60–100% neurons as 3. All brain regions were independently analyzed by three pathologists, with discrepancies resolved by consensus.   The differences in median HE and anti-P scores among different brain locations were analyzed using the Kruskal-Wallis H Test. Post-hoc multiple comparisons were performed using Dunn’s test with Bonferroni correction. All statistical analyses were performed using JMP Pro 16 software (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at P <0.05.   Clinical characteristics of the dogs Thirty-two dogs were found dead, 12 died of natural causes (illness), 23 were euthanized, and 3 died of an unknown cause. There were 31 males, 23 females, and 16 of unknown sex. The mean age of the 54 dogs (males + females) was 1.89 ± 2.59 years (range: 1 month–10 years), with 16 dogs of unknown age. Three dogs had a vaccination history, 37 had no history, and 30 had no relevant information. Twenty-three were domestic dogs, 17 were free-roaming, 11 were strays, and 19 were unknown. Major neurological symptoms were observed in 36 of the 70 dogs, including unprovoked aggressiveness, excessive drooling, mad biting at inanimate objects, paralysis, and aimless running. Non-specific clinical symptoms, such as decreased appetite, weakness, and restlessness, were observed in 13 of the 70 dogs. Data on the clinical signs of the remaining dogs were unavailable. dFAT results of Ammon’s horn and medulla tissues All Ammon’s horn and medulla specimens of the 70 dogs were positive for the rabies virus antigen in the dFAT . Histopathological findings of rabid dogs Each dog had four anatomical regions for HE and IHC examination: the cerebrum, hippocampus, thalamus, and brainstem. There were no cases in which the HE section revealed no lesions. The presence or absence of Negri bodies and the semi-quantitative results of the HE and IHC examinations for individual cases are summarized in . Microscopic findings of the rabid dog brain frequently revealed non-suppurative encephalitis consisting of Negri bodies, mononuclear perivascular cuffing, glial nodules with limited neuronal degeneration, and neuronophagia in a few cases. Eosinophilic intracytoplasmic inclusion bodies (Negri bodies) were single to a few, variable in diameter up to 8 µm, well-demarcated, round to oval in shape in pyramidal cells of the cerebrum and hippocampus . The periphery of these inclusions was heavily stained with eosin and weakly basophilic vacuoles of varied sizes (inner bodies) were observed in the center of inclusions ( , inset). These inclusions were rarely found in dendrites. Contrarily, in large neurons of the thalamus, Negri bodies were small, less than 1 μm, and less clearly demarcated from the surrounding cell cytoplasm . In the brainstem, Negri bodies were indistinct and could not be identified under high magnification with an optical microscope. Negri bodies were detected in 88.57% (62/70) of the dogs when at least one anatomical region presented an inclusion. The highest prevalence for inclusion was found in the hippocampus (61/70, 87.14%), followed by the cerebrum (49/70, 70%) and thalamus (2/70, 2.86%). There was no detection of Negri bodies in the brainstem. The prevalence for perivascular cuffing  was 75.71% (53/70) when at least one anatomical region presented perivascular cuffing with predominantly mononuclear cells. The highest occurrence (48/70, 68.57%) was found in the brainstem, followed by the thalamus (37/70, 52.86%), hippocampus (10/70, 14.29%), and the cerebrum (6/70, 8.57%). There were statistically significant differences in the mean rank of the perivascular scores between anatomical regions (χ 2 =79.25, P <0.0001). Post-hoc analysis revealed that the cerebrum (mean rank=101.96) and hippocampus (106.52) had a significantly lower score than that of the thalamus (167.56) (both P <0.0001) and brainstem (185.96) (both P <0.0001). The prevalence for glial nodule detection  was 65.71% (46/70) when at least one anatomical region presented glial nodules. The highest occurrence was found in the brainstem (38/70, 54.29%), followed by the thalamus (22/70, 31.43%), hippocampus (13/70, 18.57%), and cerebrum (6/70, 8.57%). There were statistically significant differences in the mean rank of the glial nodule scores between anatomical regions (χ 2 =40.06, P <0.0001). Post-hoc analysis revealed that the cerebrum (mean rank=112.79) had a significantly lower score than the thalamus (146.69) ( P =0.01). The brainstem (176.63) showed a significantly higher score than the thalamus ( P <0.05), cerebrum ( P <0.0001), and hippocampus (125.89) ( P <0.0001). Overall, the brainstem showed the highest scores in the rabies-positive cases. Immunohistochemical findings of rabid dogs All dFAT-confirmed rabies cases were confirmed using IHC anti-P (100%) in at least one positive anatomical region. This result indicates that the sensitivity of the IHC method was 100%. The immunoreactivity was 100% (70/70) in the hippocampus, thalamus, and brainstem and 97.14% (68/70) in the cerebrum. The mean rank of the percentage of immunoreactive cells was not statistically significantly different between anatomical regions (χ 2 =7.75, P >0.05). Rabies viral antigens were frequently visible as single to a few round to oval brown homogeneous masses in the perikaryon of the pyramidal neurons of the cerebrum and hippocampus . In contrast, there were several dozen brown granules per cell in the perikaryon of the large neurons in the thalamus  and brainstem. In a few cases, irregularly shaped homogeneous masses were observed in the thalamus and brainstem. Antigens were also detected as fine granular patterns and dust-like granules in the neuronal processes. The infiltration of inflammatory cells into the Virchow-Robin space varied from monolayer to multilayer. Although variations in the number of positive cells were observed, inflammatory cells were positive for CD3 and CD20, respectively . Mild inflammatory cell infiltration into the brain parenchyma was observed. The proliferating microglial cells, so-called glial nodules, were composed primarily of Iba1-positive cells .   Thirty-two dogs were found dead, 12 died of natural causes (illness), 23 were euthanized, and 3 died of an unknown cause. There were 31 males, 23 females, and 16 of unknown sex. The mean age of the 54 dogs (males + females) was 1.89 ± 2.59 years (range: 1 month–10 years), with 16 dogs of unknown age. Three dogs had a vaccination history, 37 had no history, and 30 had no relevant information. Twenty-three were domestic dogs, 17 were free-roaming, 11 were strays, and 19 were unknown. Major neurological symptoms were observed in 36 of the 70 dogs, including unprovoked aggressiveness, excessive drooling, mad biting at inanimate objects, paralysis, and aimless running. Non-specific clinical symptoms, such as decreased appetite, weakness, and restlessness, were observed in 13 of the 70 dogs. Data on the clinical signs of the remaining dogs were unavailable.   All Ammon’s horn and medulla specimens of the 70 dogs were positive for the rabies virus antigen in the dFAT .   Each dog had four anatomical regions for HE and IHC examination: the cerebrum, hippocampus, thalamus, and brainstem. There were no cases in which the HE section revealed no lesions. The presence or absence of Negri bodies and the semi-quantitative results of the HE and IHC examinations for individual cases are summarized in . Microscopic findings of the rabid dog brain frequently revealed non-suppurative encephalitis consisting of Negri bodies, mononuclear perivascular cuffing, glial nodules with limited neuronal degeneration, and neuronophagia in a few cases. Eosinophilic intracytoplasmic inclusion bodies (Negri bodies) were single to a few, variable in diameter up to 8 µm, well-demarcated, round to oval in shape in pyramidal cells of the cerebrum and hippocampus . The periphery of these inclusions was heavily stained with eosin and weakly basophilic vacuoles of varied sizes (inner bodies) were observed in the center of inclusions ( , inset). These inclusions were rarely found in dendrites. Contrarily, in large neurons of the thalamus, Negri bodies were small, less than 1 μm, and less clearly demarcated from the surrounding cell cytoplasm . In the brainstem, Negri bodies were indistinct and could not be identified under high magnification with an optical microscope. Negri bodies were detected in 88.57% (62/70) of the dogs when at least one anatomical region presented an inclusion. The highest prevalence for inclusion was found in the hippocampus (61/70, 87.14%), followed by the cerebrum (49/70, 70%) and thalamus (2/70, 2.86%). There was no detection of Negri bodies in the brainstem. The prevalence for perivascular cuffing  was 75.71% (53/70) when at least one anatomical region presented perivascular cuffing with predominantly mononuclear cells. The highest occurrence (48/70, 68.57%) was found in the brainstem, followed by the thalamus (37/70, 52.86%), hippocampus (10/70, 14.29%), and the cerebrum (6/70, 8.57%). There were statistically significant differences in the mean rank of the perivascular scores between anatomical regions (χ 2 =79.25, P <0.0001). Post-hoc analysis revealed that the cerebrum (mean rank=101.96) and hippocampus (106.52) had a significantly lower score than that of the thalamus (167.56) (both P <0.0001) and brainstem (185.96) (both P <0.0001). The prevalence for glial nodule detection  was 65.71% (46/70) when at least one anatomical region presented glial nodules. The highest occurrence was found in the brainstem (38/70, 54.29%), followed by the thalamus (22/70, 31.43%), hippocampus (13/70, 18.57%), and cerebrum (6/70, 8.57%). There were statistically significant differences in the mean rank of the glial nodule scores between anatomical regions (χ 2 =40.06, P <0.0001). Post-hoc analysis revealed that the cerebrum (mean rank=112.79) had a significantly lower score than the thalamus (146.69) ( P =0.01). The brainstem (176.63) showed a significantly higher score than the thalamus ( P <0.05), cerebrum ( P <0.0001), and hippocampus (125.89) ( P <0.0001). Overall, the brainstem showed the highest scores in the rabies-positive cases.   All dFAT-confirmed rabies cases were confirmed using IHC anti-P (100%) in at least one positive anatomical region. This result indicates that the sensitivity of the IHC method was 100%. The immunoreactivity was 100% (70/70) in the hippocampus, thalamus, and brainstem and 97.14% (68/70) in the cerebrum. The mean rank of the percentage of immunoreactive cells was not statistically significantly different between anatomical regions (χ 2 =7.75, P >0.05). Rabies viral antigens were frequently visible as single to a few round to oval brown homogeneous masses in the perikaryon of the pyramidal neurons of the cerebrum and hippocampus . In contrast, there were several dozen brown granules per cell in the perikaryon of the large neurons in the thalamus  and brainstem. In a few cases, irregularly shaped homogeneous masses were observed in the thalamus and brainstem. Antigens were also detected as fine granular patterns and dust-like granules in the neuronal processes. The infiltration of inflammatory cells into the Virchow-Robin space varied from monolayer to multilayer. Although variations in the number of positive cells were observed, inflammatory cells were positive for CD3 and CD20, respectively . Mild inflammatory cell infiltration into the brain parenchyma was observed. The proliferating microglial cells, so-called glial nodules, were composed primarily of Iba1-positive cells .   The present study analyzed the neuropathology in different brain regions of 70 rabid dogs in the Philippines. The percentage of Negri bodies observed in most of the dogs (88.57%) was higher than reported in Turkey, Sri Lanka, South Africa, Jordan, and previous studies on other infected animals . Consistent with findings from other countries, the Negri body was notably detected in the hippocampus . However, inclusions commonly found in the brainstem were not observed in our study . In the present study, Negri bodies demonstrated pleomorphism, ranging from a few large round to oval inclusions that were easily observable in the cerebrum and hippocampus. Occasionally, inner body-like basophilic vacuoles were observed inside the inclusions. Inversely, Negri bodies in the thalamus neurons presented small, fine, granular shapes without basophilic vacuoles, and these morphological findings were similar to lyssa bodies. Lyssa bodies are known as small aggregates of virions and matrix material without inner bodies and are undetectable by light microscopy in neuronal perikarya and dendrites . Light microscopy was unable to detect the Negri body in brainstem neurons in this study; nevertheless, the IHC results showed much more extensive distribution in infected neurons. The cause of the variable size, number, and shape of Negri bodies in different brain regions remains unknown but may be influenced partly by natural infection mode. Caudal-level nerve nuclei in the brainstem and thalamus are mainly intermediate neurons, and neurons in these areas are infected before higher neurons in the hippocampus and cerebral pyramidal neurons during retrograde infection. Therefore, it is possible that inflammatory cells infiltrate during the early stage of infection in the brainstem and thalamus, and that the early immune response inhibits virus replication and virus protein synthesis. More advanced inflammation in the lower brains likely leads to the destruction and obscuration of Negri bodies. The effect of lower brain inflammation could manifest before the upper brain lesion has fully developed. Indeed, our results indicated that the prevalence of Negri bodies was inversely proportional to the degree of inflammation, similar to the experimental dogs . The differences in the number and distribution of cytoplasmic organelles, which are essential for negrigenesis, between neurons in different brain locations may play a role. The Negri bodies represent viral factories  or might be cellular defense mechanisms . This compartmentalization, demarcated from the surrounding cytoplasm, may be an escape strategy employed by the virus to evade cell-intrinsic defense during replication and assembly, or it might represent a protective strategy by the neuron to degrade viral component, both involving aggresome pathway . In cases where Negri bodies were not observed in any area of the brain, several factors may have contributed to this finding. The stage of rabies infection could impact the visibility of Negri bodies. During the early stages, Negri bodies may not yet have formed, whereas in later stages, they might be cleared or obscured by immunity and inflammation. Additionally, the immune response of the individual hosts could play a crucial role in the detectability of Negri bodies as an aggressive immune response may inhibit their formation or lead to their destruction. Further investigation using an electron microscope is required to examine the ultrastructural differences between Negri bodies—such as large round to oval inclusions observed in the cerebrum and hippocampus, and lyssa bodies—such as small, fine, granular inclusions found in the thalamus and brainstem. The anatomical distribution of inflammation is relevant to pathogenesis. In this study, perivascular cuffing and gliosis in the cerebrum and hippocampus, where Negri bodies were more frequently detected, were generally mild or absent. In contrast, perivascular cuffing and microglial nodules were more prominent and frequently observed in the brainstem. As previously stated, it is proposed that the natural course of the virus results in more pronounced inflammation in the lower brain . Our results are partially similar to those of naturally infected rabid dogs from Thailand and India . No significant differences were observed between the cerebrum, hippocampus, and brainstem in the study of canine series from Sri Lanka . Our percentage of cases with perivascular cuffing and gliosis was higher compared to that of the study from South Africa . The differences in brain inflammation were demonstrated between two virus strains in an experimental study on rabies-infected dogs . These results indicated that the degree of perivascular inflammation and microglial cell reactivity was possibly affected by the strain type. Other factors such as study design, clinical form, and infection stage variation factors should be considered. The immunohistochemical viral antigen detection exhibited 100% sensitivity in the hippocampus, thalamus, and brainstem, matching the sensitivity of dFAT. It confirmed the diagnosis when the Negri bodies could not be seen. Several animal species from Germany showed the amounts of antigen were highest in the hippocampus, diencephalon, and tegmental part of the brain stem . In contrast, the studies from Sri Lanka, South Africa, Thailand, and India, showed that the brainstem demonstrated a higher viral antigen positivity, immunoreactivity score, or antigen distribution than that of other areas  or the others from Turkey, India, and the USA reported that the hippocampus or cerebellum or both contained the most intense signal . In our two cases of the cerebrum (cases No. 2 and 3), the tests for the rabies antigen yielded negative results. The IHC was conducted on the same slides for the cerebrum, hippocampus, and thalamus. Notably, although the hippocampus and thalamus exhibited positive immunoreactivity, the cerebrum did not. This discrepancy could be attributed to tissue decomposition, which was confirmed using HE stains and significantly impacted the diagnostic results, contrasting with Sri Lanka studies . The factors aforementioned in the inflammation section, differences in sampling and processing methods, and the degree of postmortem changes and inflammation should be considered owing to the variations in IHC results. Additionally, inconsistencies may result from studies that include species other than domestic dogs. According to the rabies virus strain, phylogenetic analysis reveals that the Philippines exclusively contains the SEA4 subclade, whereas Sri Lanka is characterized solely by the Indian subcontinent clade. SEA3 is primarily found in Thailand, and the Indian subcontinent and AL1 subclades are predominant in India . In Turkey, Jordan, Georgia, South Africa, and Europe, the virus mainly belongs to the different clades from the Philippines . Our pathological findings suggest that the observation of inclusion bodies in the hippocampus and cerebral cortex during histopathological examination could facilitate diagnosis. However, we support the suggestions from other countries  because using IHC on the brainstem is a valuable method for practical rabies diagnosis, which offers high sensitivity comparable to dFAT. This is particularly advantageous in countries where cold chain transportation is impractical and composite sampling in the field is unattainable. Using the brainstem instead of deep internal brain tissues facilitates easy sample collection and reduces hazards for practitioners by avoiding the need to access and sample deeper brain areas compared to that of other techniques.   The authors declare no conflict of interest in the present study.   