Histopathological Lung Lesions Research Articles

A study on selected responses and immune structures of broiler chickens with experimental colibacillosis with or without florfenicol administration

BackgroundColibacillosis in broiler chickens is associated with economic loss and localized or systemic infection. Usually, the last resort is antibacterial therapy. Insight into the disease pathogenesis, host responses and plausible immunomodulatory effects of the antibacterials is important in choosing antibacterial agent and optimization of the treatment. Selected responses of broiler chickens experimentally infected with Escherichia coli (E. coli) and also those treated with florfenicol are evaluated in this study. Chickens (n = 70, 5 weeks old) were randomly assigned to four groups. The control groups included normal control (NC) and intratracheal infection control (ITC) (received sterile bacterial medium). The experimental groups consisted of intratracheal infection (IT) that received bacterial suspension and intratracheal infection with florfenicol administration (ITF) group.ResultsFlorfenicol reversed the decreased albumin/globulin ratio to the level of control groups (p > 0.05). Serum interleukin 10 (IL-10) and interferon‐gamma (IFN-γ) concentrations decreased in IT birds as compared to NC group. Florfenicol decreased the serum interleukin 6 (IL-6) concentration as compared to IT group. Milder signs of inflammation, septicemia, and left shift were observed in the leukogram of the ITF group. Florfenicol decreased the severity of histopathological lesions in lungs and liver. Depletion of lymphoid tissue was detected in spleen, thymus and bursa of IT group but was absent in ITF birds. The number of colony forming units of E. coli in liver samples of ITF group was only slightly lower than IT birds.ConclusionsExperimental E. coli infection of chickens by intratracheal route is associated with remarkable inflammatory responses as shown by changes in biochemical and hematological parameters. Histopathological lesions in lymphoid organs (especially in the spleen) were also prominent. Florfenicol has positive immunomodulatory effects and improves many of the lesions before the full manifestation of its antibacterial effects. These effects of florfenicol should be considered in pharmacotherapy decision-making process.

Caracterização molecular e patologia de isolados de campo do vírus da febre aftosa em camundongos albinos suíços

Foot-and-mouth disease is responsible for severe economic losses to the livestock industry of Pakistan. This study aimed to use Swiss albino mice as a cost-effective experimental animal model to study different immunological and histopathological aspects of FMDV instead of natural targeted species like cattle. After isolation of field isolates FMDV on BHK-21 cell line, biological titer of the virus and mice infectious dose50 was calculated. Virus was injected in 45 Swiss albino mice (group A) through intraperitoneal route. The gross, histopathological and immunopathological lesions in heart, trachea and lungs were recorded at different day's intervals. Histopathologically, the heart showed congestion, hemorrhages and necrosis of cardiac muscles. Trachea showed deciliated epithelium and lungs showed hemorrhages, bronchial edema and alveolar emphysema. Immunohistochemical studies revealed the presence of virus in cardiac muscles, tracheal and bronchial epithelium and alveolar lumen. The findings evoked a thought that laboratory animals could be an alternative to large animals to meet budget limitations for further research on foot-and-mouth-disease.

Histopathological pulmonary lesions in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques experimentally infected with wild-type severe acute respiratory syndrome coronavirus 2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a viral pneumonia characterized by acute interstitial pneumonia and diffuse alveolar damage in humans. Non-human primates (NHPs) are widely used as preclinical animal models for vaccine development against SARS-CoV-2. However, the pathological changes in NHPs have been described only in selected facets and inconsistent nomenclature is used, making it difficult to interpret and compare the outcomes between studies. Here, we present a standardized methodology for histopathological evaluation of experimental infection outcomes in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques. Evaluation criteria for vascular and epithelial changes in the early (3 days post infection [dpi]) and late (21 dpi) phases of the infection were developed, and a four-grade classification encompassing all the histopathological lung lesions was established. The grades of histopathological lung lesions were higher at 3 dpi compared with 21 dpi in both species of macaques, and there were no statistically significant differences in the grades between the two species at 3 dpi and 21 dpi. This study contextualized the pathological SARS-CoV-2 presentation and standardized the terminology and grading scale for lesion severity to facilitate histopathological examination in the macaque model. By referring to the standardized histopathological criteria and grades proposed here, comparable results with high reproducibility can be obtained in future studies of pathogenicity.

Intranasal administration of a single dose of MVA-based vaccine candidates against COVID-19 induced local and systemic immune responses and protects mice from a lethal SARS-CoV-2 infection

Current coronavirus disease-19 (COVID-19) vaccines are administered by the intramuscular route, but this vaccine administration failed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infection in the upper respiratory tract, mainly due to the absence of virus-specific mucosal immune responses. It is hypothesized that intranasal (IN) vaccination could induce both mucosal and systemic immune responses that blocked SARS-CoV-2 transmission and COVID-19 progression. Here, we evaluated in mice IN administration of three modified vaccinia virus Ankara (MVA)-based vaccine candidates expressing the SARS-CoV-2 spike (S) protein, either the full-length native S or a prefusion-stabilized [S(3P)] protein; SARS-CoV-2-specific immune responses and efficacy were determined after a single IN vaccine application. Results showed that in C57BL/6 mice, MVA-based vaccine candidates elicited S-specific IgG and IgA antibodies in serum and bronchoalveolar lavages, respectively, and neutralizing antibodies against parental and SARS-CoV-2 variants of concern (VoC), with MVA-S(3P) being the most immunogenic vaccine candidate. IN vaccine administration also induced polyfunctional S-specific Th1-skewed CD4+ and cytotoxic CD8+ T-cell immune responses locally (in lungs and bronchoalveolar lymph nodes) or systemically (in spleen). Remarkably, a single IN vaccine dose protected susceptible K18-hACE2 transgenic mice from morbidity and mortality caused by SARS-CoV-2 infection, with MVA-S(3P) being the most effective candidate. Infectious SARS-CoV-2 viruses were undetectable in lungs and nasal washes, correlating with high titers of S-specific IgGs and neutralizing antibodies against parental SARS-CoV-2 and several VoC. Moreover, low histopathological lung lesions and low levels of pro-inflammatory cytokines in lungs and nasal washes were detected in vaccinated animals. These results demonstrated that a single IN inoculation of our MVA-based vaccine candidates induced potent immune responses, either locally or systemically, and protected animal models from COVID-19. These results also identified an effective vaccine administration route to induce mucosal immunity that should prevent SARS-CoV-2 host-to-host transmission.

Pathological study and detection of Bovine parainfluenza 3 virus in pneumonic sheep lungs using direct immunofluorescence antibody technique.

Bovine parainfluenza 3 virus is a common virus that causes respiratory tract infection in cattle, sheep, and goats worldwide. The objective of this study is to identify macroscopic and histopathological lung lesions in slaughtered sheep during the period from December 2018 to December 2019 and to determine the presence of Bovine parainfluenza 3 virus (BPI3V) in frozen sheep pneumonic lung using a direct immunofluorescence antibody technique (DFAT). The overall prevalence of lung affection was 11% (1440/13084). The gross lesions were acute bronchopneumonia (58.12%), interstitial pneumonia (07.15%), fibrinous bronchopneumonia (10.70%), suppurative bronchopneumonia (03.47%), verminous pneumonia (13.75%), and ovine pulmonary adenomatosis (06.81%). There was a significant difference in the rate of pulmonary lesions according to the seasons of the study. The lesions were more frequently observed in autumn and winter with a rate of 34.17% and 28.05%, respectively. The DFAT was carried out only on 107 pneumonic samples with interstitial pneumonia, fibrinous bronchopneumonia, and acute bronchopneumonia. The BPI3V antigens were detected in 12 samples (11.21%). This is the first study that revealed the presence of the BPI3V in pneumonic sheep lungs in Batna region using the direct immunofluorescence antibody technique. The latter may be used for definite diagnosis when histopathological modifications in pneumonic sheep caused by this virus are difficult to distinguish from those caused by other respiratory viruses.

Lesions in the lungs of fatal corona virus disease Covid-19

The corona virus outbreak in Wuhan, China, at the end of 2019 has rapidly evolved into a pandemic which is still virulent in many countries. An infection with SARS-CoV-2 can lead to corona virus disease (Covid-19). This paper presents an overview of the knowledge gained so far with regard to histopathological lung lesions in fatal courses of Covid-19. The main findings were diffuse alveolar damage and micro-angiopathies. These included the development of hyaline membranes, thrombi, endothelial inflammation, haemorrhages and angiogenesis. Overall, the vessel lesions seemed to be more lethal than the diffuse alveolar damage. There was obvious hyperreactivity and hyperinflammation of the cellular immune system. An expanded T-cell memory may explain the increased risk of a severe course in the elderly.

Differential Viral-Host Immune Interactions Associated with Oseltamivir-Resistant H275Y and Wild-Type H1N1 A(pdm09) Influenza Virus Pathogenicity.

Oseltamivir is a common therapy against influenza A virus (IAV) infections. The acquisition of oseltamivir resistance (OR) mutations, such as H275Y, hampers viral fitness. However, OR H1N1 viruses have demonstrated the ability to spread throughout different populations. The objective of this work was to compare the fitness of two strains of OR (R6 and R7) containing the H275Y mutation, and a wild-type (F) pandemic influenza A (H1N1) 2009 (pdm09) virus both in vitro and in vivo in mice and to select one OR strain for a comparison with F in ferrets. R6 showed faster replication and pathogenicity than R7 in vitro and in mice. Subsequently, R6 was selected for the fitness comparison with the F strain in ferrets. Ferrets infected with the F virus showed more severe clinical signs, histopathological lung lesions, and viral quantification when compared to OR R6-infected animals. More importantly, differential viral kinetics correlated with differential pro-inflammatory host immune responses in the lungs of infected ferrets, where OR-infected animals developed a protective higher expression of type I IFN and Retinoid acid Inducible Gene I (RIG-I) genes early after infection, resulting in the development of milder disease. These results suggest the presence of early specific viral-host immune interactions relevant in the development of influenza-associated lung pathology.

Pulmonary lesions in slaughtered sheepin Western Iran: gross and histopathological findings.

This study was conducted with the objective of identifying the types of gross and histopathological lesions in lungs of sheep slaughtered between March 2013 to February 2014 at Kermanshah abattoir, west of Iran. A total of 1200 slaughtered sheep lungs was inspected during the study period. Pulmonary lesions were detected in 445 (37.08%) carcasses, grossly. Lesions were further subjected for detail histopathological examinations. On microscopic examination, most lungs had more than one type of lesions and thirteen different lesions were observed. The most common lesions were bronchopneumonia (13.33%), pulmonary emphysema (4.25%), hydatid cyst (4.16%), interstitial pneumonia (3.58%), abscess (3.33%), atelectasis (2.83), Pleurisy (1.66), bronchiectasis (1.41), verminous pneumonia (0.91%) and pulmonary adenomatosis (0.25%). The highest prevalence of pulmonary lesions was observed in the winter, which differences were significant in compared to other seasons (P=0.007). This study demonstrates that lung diseases and lesions represent a serious problem and may continue to be a drawback to livestock industry and may pose health risks to meat consumers from contrasting zoonotic diseases in western Iran. So designing of serious prevention and treatment programs are very important in our region.

Validity of genus Perostrongylus Schlegel, 1934 with new data on Perostrongylus falciformis (Schlegel, 1933) in European badgers, Meles meles (Linnaeus, 1758): distribution, life-cycle and pathology

BackgroundA century of debates on the taxonomy of members of the Metastrongyloidea Molin, 1861 led to many reclassifications. Considering the inconstant genus assignation and lack of genetic data, the main aim of this study was to support the validity of the genus Perostrongylus Schlegel, 1934, previously considered a synonym of Aelurostrongylus Cameron, 1927, based on new molecular phylogenetic data and to understand its evolutionary relationships with other metastrongyloid nematodes.ResultsSpecimens of lungworm collected from European badgers in Germany, Romania and Bosnia and Herzegovina were morphologically and molecularly (rDNA, cox1) characterized. From a phylogenetic standpoint, Perostrongylus is grouped with high support together with the genera Filaroides van Beneden, 1858 and Parafilaroides Dougherty, 1946 and includes probably two species: Perostrongylus falciformis (Schlegel, 1933), a parasite of Meles meles in Europe and P. pridhami (Anderson, 1962), a parasite of Neovison vison in North America. Perostrongylus and Aelurostrongylus are assigned to different clades. Aelurostrongylus becomes a monotypic genus, with the only species Aelurostrongylus abstrusus (Railliet, 1898). In addition, we provide morphological and morphometric data for the first-stage (L1), second-stage (L2), and third-stage (L3) larvae of P. falciformis and describe their development in experimentally infected Cornu aspersum snails. The pathological and histopathological lesions in lungs of infected European badgers are also described. This is the first record of P. falciformis in Romania.ConclusionsMolecular phylogenetic and morphological data support the validity of the genus Perostrongylus, most probably with two species, P. falciformis in European badgers and P. pridhami in minks in North America. The two genera clearly belong to two different clades: Perostrongylus is grouped together with the genera Filaroides and Parafilaroides (both in the family Filaroididae Schulz, 1951), whereas Aelurostrongylus belongs to a clade with no sister groups.

Emergence of a novel highly pathogenic recombinant virus from three lineages of porcine reproductive and respiratory syndrome virus 2 in China 2017.

A novel porcine reproductive and respiratory syndrome virus 2 (PRRSV2) was isolated from diseased piglets in Shandong, China in 2017 and denominated as SD17-38. ORF5 sequencing showed that SD17-38 contains a unique serine/asparagine deletion at position 33 and an asparagine insertion at position 60 of GP5, which has never been described. The SD17-38 complete genome was then determined, and genome-based phylogenetic analysis showed that SD17-38 is clustered with NADC30-like isolates. Sequence alignment and recombination analyses by RDP4 and SimPlot all indicated that SD17-38 is a recombinant virus from NADC30 (lineage 1), BJ-4 (lineage 5) and TJ (lineage 8) isolates. Animal challenge study in 4-week piglets showed that SD17-38 causes high fever (≥41°C), 100% morbidity and 40% mortality. In addition, significantly lower weight gain and severe histopathological lung lesions could be observed in SD17-38-infected pigs. In particular, the unique deletion and insertion in GP5 were stable during the challenge study. This study provides direct evidence for the natural occurrence of recombination events among three lineages of PRRSV2 in Chinese swine herds, resulting in the emergence of novel PRRSV variant with unique genetic property and high pathogenicity.

English

Animals in sub-Saharan African countries are used as source of milk, meat, hides and skin where animal diseases are one of the primary constraints in increasing the productivity of food animals. Studies were conducted to examine various respiratory problems of cattle with purpose of identifying the gross and histopathological lesions in lungs of slaughtered cattle at Abergelle Export Abattoir from April 2015 to June 2015 Mekelle, Tigray, Ethiopia. A total of 240 lungs of cattle originating from different parts of Tigray regions were examined and as many as 208 lung samples with gross pulmonary lesions were collected for further confirmatory histopathological study. The prevalence of pulmonary lesions of cattle in this study was 86.6% (n=208). The case of pneumonia was found to be highest followed by lung emphysema and congestion besides cases of oedema, hydatidosis, abscess and atelectasis. These results indicated that respiratory problem is still a major health problem in cattle production. Key words: Lung, pneumonia, pulmonary lesion, Ethiopia.

An Improved Tobacco Mosaic Virus (TMV)-Conjugated Multiantigen Subunit Vaccine Against Respiratory Tularemia.

Francisella tularensis, the causative agent of the fatal human disease known as tularemia is classified as a Category A Select Agent by the Centers for Disease Control. No licensed vaccine is currently available for prevention of tularemia in the United States. Previously, we published that a tri-antigen tobacco mosaic virus (TMV) vaccine confers 50% protection in immunized mice against respiratory tularemia caused by F. tularensis. In this study, we refined the TMV-vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetra-antigen vaccine by conjugating OmpA, DnaK, Tul4, and SucB proteins of Francisella to TMV. CpG was also included in the vaccine formulation as an adjuvant. Primary intranasal (i.n.) immunization followed by two booster immunizations with the tetra-antigen TMV vaccine protected 100% mice against i.n. 10LD100 challenges dose of F. tularensis live vaccine strain (LVS). Mice receiving three immunization doses of tetra-antigen TMV vaccine showed only transient body weight loss, cleared the infection rapidly, and showed minimal histopathological lesions in lungs, liver, and spleen following a lethal respiratory challenge with F. tularensis LVS. Mice immunized with the tetra-antigen TMV vaccine also induced strong ex vivo recall responses and were protected against a lethal challenge as late as 163 days post-primary immunization. Three immunization with the tetra-antigen TMV vaccine also induced a stronger humoral immune response predominated by IgG1, IgG2b, and IgG2c antibodies than mice receiving only a single or two immunizations. Remarkably, a single dose protected 40% of mice, while two doses protected 80% of mice from lethal pathogen challenge. Immunization of Interferon-gamma (IFN-γ)-deficient mice with the tetra-antigen TMV vaccine demonstrated an absolute requirement of IFN-γ for the generation of protective immune response against a lethal respiratory challenge with F. tularensis LVS. Collectively, this study further demonstrates the feasibility of TMV as an efficient platform for the delivery of multiple F. tularensis antigens and that tetra-antigen TMV vaccine formulation provides complete protection, and induces long-lasting protective and memory immune responses against respiratory tularemia caused by F. tularensis LVS.

Guinea pig infected with Mycobacterium tuberculosis via oral consumption

ABSTRACTGuinea pig represents a highly suitable animal model for study of Mycobacterium tuberculosis(Mtb)infection, as it demonstrates similarities to Mtb pulmonary infection in humans. It is known that guinea pigs can be efficiently infected by the respiratory or subcutaneous exposure to Mtb. However, information on Mtb infection through oral route is almost absent in the literature. Here, we examined whether guinea pigs can be infected by drinking Mtb containing water. Our findings confirmed that the guinea pigs could be infected with Mtb via drinking virulent water. The infected guinea pigs developed uniform oval-craterform ulcers at the 30th day after infection. Bacterial cultures showed Mtb growth in the lungs and spleens from the guinea pigs infected with high dose of Mtb. In addition, the infected animals had histopathological granulomatous lesions in lungs, spleens and mesenteric lymph nodes. We provide the compelling evidence that the guinea pigs could be infected by drinking Mtb containing water. The clinical and pathological observations in the infected animals were similar to those found in guinea pigs infected via the respiratory or subcutaneous routes.

Recombination in JXA1-R vaccine and NADC30-like strain of porcine reproductive and respiratory syndrome viruses

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most devastating swine diseases worldwide, resulting in immense economic losses. PRRS virus (PRRSV) has undergone rapid evolution since its first recognition in 1990s. In the present study, a PRRSV strain named FJXS15 causing high morbidity and mortality was isolated from piglets and sows from a farm participating in vaccination in China. Phylogenetic and molecular evolutionary analyses revealed that FJXS15 was highly similar to the JXA1-R vaccine strain (a live attenuated virus vaccine strain derived from the highly pathogenic PRRSV JXA1) in the ORF1a (nt 901-)−ORF4 (-nt 419) coding regions, as well as to FJZ03 (lineage 1, NADC30-like) in the 5′-UTR, ORF5a−ORF7 coding regions, and 3′-UTR, suggestive of a natural recombination event. Recombination analyses showed that recombination events occurred in two inter-lineage recombination events between Lineages 1 and 8 based on based on classification system (Shi et al., 2010), and two recombination breakpoints at positions 1–1092 and 13771–15537 of the sequence alignment (with reference to the VR-2332 strain). Animal experiments demonstrated that FJXS15-infected animals had more severe histopathological lung lesions than did JXA1-R-infected and control groups. A 25% mortality rate was found in FJXS15-infected piglets, which was similar to that found with other Chinese HP-PRRSV strains. Thus, the recombinant virus is a highly virulent PRRSV. Moreover, this report provides evidence for inter-subgenotypic recombination between the JXA1-R vaccine virus and a circulating Lineage 1 virus.

Emergence of a novel highly pathogenic porcine reproductive and respiratory syndrome virus in China.

From 2014 to 2015, four novel highly pathogenic PRRS virus (HP-PRRSV) strains named 14LY01-FJ, 14LY02-FJ 15LY01-FJ, and 15LY02-FJ were isolated from high morbidity (100%) and mortality (40%-80%) in piglets and sows in Fujian Province. To further our knowledge about these novel virus strains, we characterized their complete genomes and determined their pathogenicity in piglets. Full-length genome sequencing analysis showed that these four isolates were closely related to type 2 (North American type, NA-type) isolates, with 88.1%-96.3% nucleotide similarity, but only 60.6%-60.8% homology to the Lelystad virus (LV) (European type, EU-type). The full length of the four isolates was determined to be 15017 or 15018 nucleotides (nt), excluding the poly(A) tail. Furthermore, the four isolates had three discontinuous deletions (aa 322-432, aa 483, and aa 504-522) within hypervariable region II (HV-II) of Nsp2, as compared to the reference strain VR-2332. This deletion pattern in the four isolates is consistent with strain MN184 and strain NADC30 isolated from America. Phylogenetic and molecular evolutionary analyses indicated that these virulent strains originated from a natural recombination event between the JXA1-like HP-PRRSV (JXA-1 is one of the earliest Chinese HP-PRRSV strains; sublineage 8.7) and the NADC30-like (lineage 1) PRRSV. Animal experiments demonstrated that these four strains caused significant weight loss and severe histopathological lung lesions as compared to the negative control group. High mortality rate (40% or 80%) was found in piglets infected with any one of the four strains, similar to that found with other Chinese HP-PRRSV strains. This study showed that the novel variant PRRSV was HP-PRRSV, and it is therefore critical to monitor PRRSV evolution in China and develop a method for controlling PRRS.

Efficacy of Mycoplasma hyopneumoniae vaccination before and at weaning against experimental challenge infection in pigs.

BackgroundCommercial bacterins are widely used at weaning to control Mycoplasma hyopneumoniae infections in pigs. However, it is not known whether the efficacy of vaccinating against M. hyopneumoniae can be influenced by the weaning process when vaccination is applied at the day of weaning. The present study assessed the efficacy of a single M. hyopneumoniae vaccination (Ingelvac MycoFLEX®) three days before weaning (V1) or at weaning (V2) against experimental challenge infection. Four weeks after vaccination, groups V1 and V2 (n = 20 pigs each) and a non-vaccinated, positive control group (PCG) (n = 20) were endotracheally inoculated with a virulent M. hyopneumoniae field strain. Five pigs were used as a negative control group. All pigs were euthanized 5 weeks after challenge. The main parameters investigated included macroscopic and histopathological lung lesions at necropsy, immunofluorescence (IF) staining and quantitative real-time PCR (qPCR) on broncho-alveolar lavage (BAL) fluid for quantifying M. hyopneumoniae.ResultsThe average macroscopic lung lesion scores in groups V1, V2 and PCG were 0.54, 0.88 and 1.04, respectively (P > 0.05). The average lymphohistiocytic infiltration scores in groups V1, V2 and PCG were 2.95, 3.16 and 3.61, respectively (P < 0.05). The average IF scores were: V1 = 1.13, V2 = 1.19 and PCG = 1.25 (P > 0.05), the qPCR values were: V1 = 102.94, V2 = 102.76 and PCG = 103.23 (P > 0.05). All pigs of the negative control group remained negative throughout the study.ConclusionsBoth vaccinated groups had lower numbers of macroscopic and histopathological lung lesions, and lower numbers of M. hyopneumoniae organisms in the BAL fluid compared to the PCG. However, no firm conclusions could be made on whether weaning negatively influences the efficacy of M. hyopneumoniae vaccination, since significant differences between the treatment groups were only obtained for the histopathological lung lesions. This could be attributed to the fact that milder macroscopic lung lesions were produced in the inoculated pigs, when compared to previous trials conducted by the same group. Further research under field conditions is warranted to assess possible differences between the two vaccination strategies.

Comparative Experimental Infection Study in Dogs with Ehrlichia canis, E. chaffeensis, Anaplasma platys and A. phagocytophilum.

Dogs acquire infections with the Anaplasmataceae family pathogens, E. canis, E. chaffeensis, E. ewingii, A. platys and A. phagocytophilum mostly during summer months when ticks are actively feeding on animals. These pathogens are also identified as causing diseases in people. Despite the long history of tick-borne diseases in dogs, much remains to be defined pertaining to the clinical and pathological outcomes of infections with these pathogens. In the current study, we performed experimental infections in dogs with E. canis, E. chaffeensis, A. platys and A. phagocytophilum. Animals were monitored for 42 days to evaluate infection-specific clinical, hematological and pathological differences. All four pathogens caused systemic persistent infections detectible throughout the 6 weeks of infection assessment. Fever was frequently detected in animals infected with E. canis, E. chaffeensis, and A. platys, but not in dogs infected with A. phagocytophilum. Hematological differences were evident in all four infected groups, although significant overlap existed between the groups. A marked reduction in packed cell volume that correlated with reduced erythrocytes and hemoglobin was observed only in E. canis infected animals. A decline in platelet numbers was common with E. canis, A. platys and A. phagocytophilum infections. Histopathological lesions in lung, liver and spleen were observed in all four groups of infected dogs; infection with E. canis had the highest pathological scores, followed by E. chaffeensis, then A. platys and A. phagocytophilum. All four pathogens induced IgG responses starting on day 7 post infection, which was predominantly comprised of IgG2 subclass antibodies. This is the first detailed investigation comparing the infection progression and host responses in dogs after inoculation with four pathogens belonging to the Anaplasmataceae family. The study revealed a significant overlap in clinical, hematological and pathological changes resulting from the infections.

Toxicity Study of Pittosporum ochrosiaefolium Bojer (Pittosporaceae) a Medicinal Plant of Madagascar

The present work aimed to assess the leaf toxicity of Pittosporum ochrosiaefolium Bojer, a well-known medicinal plant endemic to Madagascar. Leaf methanolic extract (LME), obtained after successive extractions by hexan and methanol, was tested in vivo on warm and cold-blooded animals and in vitro on isolated atria of guinea-pig. LME was toxic to mice with a LD50 of about 46.69 mg/kg of body weight by intraperitoneal route. It induced mainly nervous disorders (body fasciculation, clonic convulsions), respiratory troubles (reduction of respiration frequency and cyanosis) and diarrheas. By intraperitoneal route, LME (46.69 mg/kg) caused histopathological lesions in lungs, liver, kidneys, small and large intestines but had no effects on brain, heart and stomach. Vascular congestion, inflammatory infiltrates, edema and necrosis were frequently observed. LME had a positive inotropic effect but no significant chronotropic one on isolated atria. It did not alter renal and hepatic functions at 21.24 mg/kg. It was highly toxic to the frog Ptychadena mascareniensis (LC50 of 13.51 µg/mL) and the fish Cyprinus carpio (LC50 of 8.2 µg/mL). It was also toxic to mosquito larvae Culex quinquefasciatus and Aedes albopictus with LC50 of 720 ppm and 910 ppm respectively. Different chemical compound groups were found in LME but only saponins proved to be toxic. Under certain conditions, P. ochrosiaefolium might be exploited as source of pesticides or therapeutic molecules.

Preclinical testing of a vaccine candidate against tularemia.

Tularemia is caused by a gram-negative, intracellular bacterial pathogen, Francisella tularensis (Ft). The history weaponization of Ft in the past has elevated concerns that it could be used as a bioweapon or an agent of bioterrorism. Since the discovery of Ft, three broad approaches adopted for tularemia vaccine development have included inactivated, live attenuated, or subunit vaccines. Shortcomings in each of these approaches have hampered the development of a suitable vaccine for prevention of tularemia. Recently, we reported an oxidant sensitive mutant of Ft LVS in putative EmrA1 (FTL_0687) secretion protein. The emrA1 mutant is highly sensitive to oxidants, attenuated for intramacrophage growth and virulence in mice. We reported that EmrA1 contributes to oxidant resistance by affecting the secretion of antioxidant enzymes SodB and KatG. This study investigated the vaccine potential of the emrA1 mutant in prevention of respiratory tularemia caused by Ft LVS and the virulent SchuS4 strain in C57BL/6 mice. We report that emrA1 mutant is safe and can be used at an intranasal (i. n.) immunization dose as high as 1x106 CFU without causing any adverse effects in immunized mice. The emrA1 mutant is cleared by vaccinated mice by day 14–21 post-immunization, induces minimal histopathological lesions in lungs, liver and spleen and a strong humoral immune response. The emrA1 mutant vaccinated mice are protected against 1000–10,000LD100 doses of i.n. Ft LVS challenge. Such a high degree of protection has not been reported earlier against respiratory challenge with Ft LVS using a single immunization dose with an attenuated mutant generated on Ft LVS background. The emrA1 mutant also provides partial protection against i.n. challenge with virulent Ft SchuS4 strain in vaccinated C57BL/6 mice. Collectively, our results further support the notion that antioxidants of Ft may serve as potential targets for development of effective vaccines for prevention of tularemia.

Aspergillosis in a rescued Himalayan griffon vultureGyps himalayenensis: A case study

A rare case of pulmonary aspergillosis was documented in a Himalayan griffon vulture with characteristic gross and histopathological lesions in lung and air sacs. Gross changes were observed in lungs and air sacs with yellowish white nodules of 0.5–3mm in diameter including thickening of air sacs. Histopathologically, multifocal caseous necrotic areas, inflammatory cellular infiltrations, presence of fungal hyphae and fibrous tissue proliferation in lungs and air sacs were observed. The characteristic hyphae of Aspergillus spp. were demonstrated in the nodules of lung in histopathological sections stained by Gridley's and H & E staining method.