This study established a modified immunohistochemistry method using murine antiserum as the primary antibody to detect mouse hepatitis virus (MHV) and Mycoplasma pulmonis antigens in formalin-fixed tissue sections. Using this method, MHV antigen was detected in the liver, stomach, caecum, colon and spleen of infected mice. Mycoplasma pulmonis antigen was demonstrated on the luminal surface of bronchioles in infected rats. This technique provides a useful method for diagnosing MHV and M. pulmonis infections when commercial antibodies are unavailable or serological diagnosis is not possible due to immunosuppression.
Paratuberculosis (PTB) remains one of the most obstacles limit animal breeding sector all over the world. The current study aimed to detect the etiology of PTB in tissues of clinically suspected small ruminants using histopathological and real-time polymerase chain reaction (RT-PCR) methods. Clinical examination showed 10 (26.4%) PTB suspected cases out of the total (38) examined animals. The suspected cases were euthanized, necropsied, gross lesions were recorded and tissue samples were collected for histopathological and molecular procedures. Grossly intestinal and mesenteric lymph nodes thickening, corrugations and edematous swellings were recorded. Semi-thin sections of the intestine and mesenteric lymph nodes stained with toluidine blue demonstrated MAP organism inside epithelium cells and macrophages. RT-PCR detected MAP IS900 gene in all suspected cases (100%), thus we recommend using RT-PCR as a rapid sensitive method in the diagnosis of PTB.
Key-words: Paratuberculosis, Mycobacterium, Semi thin sections, Toluidine blue, IS900 gene
Antiplasmodial activity of methanol leaf extract of Salacia senegalensis Lam ...Premier Publishers
The antiplasmodial effect of methanol leaf extract of Salacia senegalensis were evaluated in albino mice infected with chloroquine-sensitive Plasmodium berghei berghei (NK65) in order to justify or otherwise its use as antimalarial remedy in Nigeria folk medicine. Activities investigated were suppressive effect against early infection, curative effect against established infection and prophylactic effect against residual infection. Results showed a dose dependent blood schizontocidal activity at all the phases of malarial infection studied. The in vivo antiplasmodial effect of the extract (1000, 1200 and 1400 mg/kg body weight) against P. berghei showed significant (p < 0.05) dose-dependent activity for suppressive, curative and prophylactic test. When the extract dose increased from 1000 to 1400 mg/kg/day, chemosuppressive activity of the extract increased from 66.47 % to 80.33 %. There was also an increase from 66.57 % to 75.41 % and from 64.90 % to 82.72 % for the repository and curative activities respectively. The schizontocidal activities were comparable to that of chloroquine -which had percentage suppression of parasitaemia as 87.03 %, 85.12 %, and 91.68 % for suppressive, prophylactic and curative activities respectively). It was thus concluded that the herbal extract possesses significant antimalarial potency which could be exploited in the formulation of antimalarial drugs.
Paratuberculosis (PTB) remains one of the most obstacles limit animal breeding sector all over the world. The current study aimed to detect the etiology of PTB in tissues of clinically suspected small ruminants using histopathological and real-time polymerase chain reaction (RT-PCR) methods. Clinical examination showed 10 (26.4%) PTB suspected cases out of the total (38) examined animals. The suspected cases were euthanized, necropsied, gross lesions were recorded and tissue samples were collected for histopathological and molecular procedures. Grossly intestinal and mesenteric lymph nodes thickening, corrugations and edematous swellings were recorded. Semi-thin sections of the intestine and mesenteric lymph nodes stained with toluidine blue demonstrated MAP organism inside epithelium cells and macrophages. RT-PCR detected MAP IS900 gene in all suspected cases (100%), thus we recommend using RT-PCR as a rapid sensitive method in the diagnosis of PTB.
Key-words: Paratuberculosis, Mycobacterium, Semi thin sections, Toluidine blue, IS900 gene
Antiplasmodial activity of methanol leaf extract of Salacia senegalensis Lam ...Premier Publishers
The antiplasmodial effect of methanol leaf extract of Salacia senegalensis were evaluated in albino mice infected with chloroquine-sensitive Plasmodium berghei berghei (NK65) in order to justify or otherwise its use as antimalarial remedy in Nigeria folk medicine. Activities investigated were suppressive effect against early infection, curative effect against established infection and prophylactic effect against residual infection. Results showed a dose dependent blood schizontocidal activity at all the phases of malarial infection studied. The in vivo antiplasmodial effect of the extract (1000, 1200 and 1400 mg/kg body weight) against P. berghei showed significant (p < 0.05) dose-dependent activity for suppressive, curative and prophylactic test. When the extract dose increased from 1000 to 1400 mg/kg/day, chemosuppressive activity of the extract increased from 66.47 % to 80.33 %. There was also an increase from 66.57 % to 75.41 % and from 64.90 % to 82.72 % for the repository and curative activities respectively. The schizontocidal activities were comparable to that of chloroquine -which had percentage suppression of parasitaemia as 87.03 %, 85.12 %, and 91.68 % for suppressive, prophylactic and curative activities respectively). It was thus concluded that the herbal extract possesses significant antimalarial potency which could be exploited in the formulation of antimalarial drugs.
Enterocin 55 produced by non rabbit-derived strain Enterococcus faecium EF55 ...Agriculture Journal IJOEAR
— Ent55 is produced by poultry strain Enterococcus faecium EF55. It is substance which can be allotted to Class II enterocins; thermo-stable, small peptide. Because producer strain has shown beneficial effect in poultry and broiler rabbits as well, we decided to apply Ent55 in broiler rabbit husbandry. Ent55 showed antimicrobial activity in broiler rabbits by reduction of staphylococci, Clostridiae, pseudomonads and coliforms. Its beneficial effect was demonstrated by stimulation of phagocytic activity as well as by reduction of Eimeria spp. oocysts. GPx values were lower; it means, no oxidative stress was evoked. Moreover, it has not negative influence on growth performance and biochemical parameters. Our results indicated that enterocin produced by not-autochtonous strain can also have protective and beneficial effect in broiler rabbits.
Protective Effect of Egyptian Propolis Against Rabbit PasteurellosisBee Healthy Farms
Propolis is known for its protective effects on humans and animals, including improving respiratory conditions. It's also documented to be a very complementary adjuvant with other treatment modalities.
Pasteurella multocida is a well known cause of morbidity and mortality in rabbits. The predominant syndrome is upper respiratory disease or "snuffles." P. multocida is often endemic in rabbit colonies and the acquisition of infection in young rabbits is correlated to the prevalence in adult rabbits.
Mobile phone has been source of microorganisms that cause diseases of public health concerns. In a study, one-fi fth of cellular phones examined were found to harbor pathogenic bacteria indicating that these devices may serve as vehicles of transmission. Swab samples were collected aseptically from the phones of different handlers like motor bike riders, food vendors, meat sellers and nursing mothers. Bacteria isolation and identifi cation were carried out using pour plating technique with distinctive morphological and biochemical characteristics.The pathogenicity of the bacterial isolates was investigated through oral inoculation into albino rats. Eighty-eight (88) bacteria were isolated and selected based on their resistance to antibiotics for pathological study. Loss in weight was observed in some albino rat. Along with reduction in the packed cell volume, hemoglobin but raised white blood cell. Animal inoculated with Bacillus cereus showed meningitis like symptom after the first week of inoculation. Also, there were short and stunted villi; low crystal depth with necrotic
debris in the lumen. It has been observed that cell phones may harbor pathogenic bacteria and can subsequently plays role as fomite in the disease transmission. Therefore, the need to educate community phone handlers in the rural area becomes imperative.
ABSTRACT- A number of 18 adults male outbred albino rats, weighing between 133-137g were used to investigate the drug susceptibility of Trypanosoma evansi strain isolated from naturally infected dromedary camels in Umbadir area, North Kordofan State, Sudan. The rats were divided into 3 groups (C, D and F) of 6 animals each. Group C and D were infected intraperitoneally with T. evansi (Umbadir stabilate) with 1×104 Trypanosome for the inoculum. Group D rats were given quinapyramine sulphate (20 mg/Kg bwt) after parasitaemia was evident. Group F was left as healthy uninfected control for the stabilate. When parasite counts were one or more parasites per field, counting in haemocytometer were used for exact number of parasite per cubic millimeter using Neubaeur’s counter. Parasites from tail blood were first fixed, stained and diluted in trypanosome diluting reagent. The parasites were diluted to the level that can be easily counted in WBC counting chamber in the haemocytometer. The total number of parasites was expressed as log10 number of parasites per ml of blood. The presence and degree of parasitaemia were determined daily for each rat by examining tail blood. The identity of the local stabilates of Trypanosoma evansi was confirmed through adopting PCR where primers that target the internal transcribed spacer one (ITS1) of the ribosomal DNA were used. There was significant reduction in serum glucose and potassium as well as significant increase in total protein, urea, calcium, albumin and cholesterol in group C. The Umbadir stabilate showed low mortality and high sensitivity to quinapyramine sulphate.
Key-words- Drug susceptibility, T. evansi, Dromedary camels, Sudan
Hepato Protective Assessment of Pawpaw Leaves, Neem, Lemon Grass and Acts on ...ijtsrd
Malaria is a major concern in Nigeria, and stands as the second leading cause of death from all infectious disease in Africa. Several studies have reported the damaging effect of the parasite to various body organs especially the liver. Reports over time has shown the benefits of various plants extracts in ethno medicine. However, not much have been done on the effects of some of these extracts in combined form on its hepato protective assessment in comparison with any known ACT based anti malaria. The focus of this study was to explore the hepato protective properties of ethanoic extract of Carica papaya Linn, AzadirachtaIndica, CymbopogonCitratusagainst ACT based antimalarial therapy on plasmodium berghei parasitized wistar rats. Phytochemical analysis of the extracts were done according to the method described by Treaseand Evans. Hepato protective assessment were done using the liver function tests and assay of the liver histology respectively. One hundred and ten 110 rats distributed into 11 groups, each group having 10rats were used for the experiment. Negative control received just feed and water, Positive control were induced with the malaria parasite and given feed and water only. The tests groups were induced with malaria, received feed and water and treated with 500mg kg, 250mg kg and 165mg kg doses of the extracts, both individually and in combined forms, as well as the standard ACT anti malaria. Phytochemical screening showed that the plant extracts possessed high concentration of Tannins, Flavonoids, Saponins and Alkaloids. Plasmodium berghei increased the activities of ALP, ASP and ALT when compared with the positive control group. This may be attributed to increase in functional capacity of the liver as a result of the presence of the infection for the tests groups. Treatment with the plant extracts decreased ALP and ALT levels significantly P 0.05 , as well as AST levels except for the Neem extract. Histological examination of the liver of test animals showed no extensive damage to the tissue by the individual extracts when compared to the negative control group. Nnyaha Anthonia E. | Igbokwe Ugochukwu V. | Okonkwo Onyeka Chukwudi | Ajeka Prisca O. | Nwaissac Ikechukwu S. | Okpa Precious N. "Hepato-Protective Assessment of Pawpaw Leaves, Neem, Lemon Grass and Acts on Plasmodium Berghei Parasitized Wistar Rats" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-5 , August 2021, URL: https://www.ijtsrd.com/papers/ijtsrd46292.pdf Paper URL: https://www.ijtsrd.com/medicine/physiology/46292/hepatoprotective-assessment-of-pawpaw-leaves-neem-lemon-grass-and-acts-on-plasmodium-berghei-parasitized-wistar-rats/nnyaha-anthonia-e
Clinical Manifestations of Plasmodium bergheiANKA Infection in Juvenile Mice:...AI Publications
Malaria is an important health and development challenge in Africa, Animalmodels most particularly mice, have long been employedto study malaria pathogenesis. Clinical manifestations due to Plasmodium bergheiANKA infection in juvenile mice as a model for understanding the complications ofcongenital malaria in neonates.Forty-five juvenile mice (5-7 days old) were acquired from University College Hospital, Ibadan and injected with 2 x 107 (0.2ml) Plasmodium berghei ANKA parasitized red blood cells (PRBCs). Mice were transported to the study site, kept in well ventilated cages and fed daily with a balanced ration. Every day after post-P. berghei infection, mice were monitored for mortality. Clinical manifestations ofexperimental cerebral malaria (ECM) was assessed and confirmed if at leastruffled fur, hunching, wobbly gait, limb paralysis, convulsions, or coma was observed. Each sign was given a score of 1. Animals with scores ≥4 were considered to have severe ECM.20 (44%) micewerelost due to natural cause (i.e. stress) at day 2 of the experiment. Between day 4 and 9, 25 (56%) of the studymice presented clinical signs of ECM which includes; ruffled fur 25(100%), hunching 21 (84%), wobbly gait 17 (68%), limb paralysis 20 (80%), convulsions 25 (100%) and subsequently died. Survival rate and severity of ECM in the mice differs, 22 (88.0%) had severe ECM and 3(12.0%) had mild ECM.This study has shown that parasite establishment and malaria complications can manifest as early as 4 days’postP. berghei infection in 5-7 days old mice.
Johne's disease (JD) or Paratuberculosis (PTB) has gained a great attention by many industrial countries for its severing economic losses and possibly zoonotic concerns. In the current study conventional clinical and direct microscopic examination compared to real time polymerase chain reaction (RT-PCR) were used to diagnose JD in clinically suspected small ruminants. Clinical examination revealed 130 (8.7%) suspected cases that showed history of emaciation and diarrhea out of the total examined (1500) animals. Direct microscopy of Ziehl-Neelsen (ZN) stained smears (130) revealed 62 (47.7%) acid fast bacteria resembled Mycobacterium avium subsp. paratuberculosis (MAP). RT-PCR insertion sequence gene (IS900) detected MAP in 25 (65.8%) out of 38 fecal samples harbored acid fast bacilli. We concluded and recommended that RT-PCR considers the most rapid confirmatory method for screening and diagnosis of the MAP in comparison to low specific conventional phenotypic methods, which still remained valuable techniques in the diagnosis of JD in developing countries.
Key-words: Johne's disease, Paratuberculosis, Acid fast bacteria, Ziehl-Neelsen stain, IS900 gene
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research paper publishing, where to publish research paper, journal publishing, how to publish research paper, Call for research paper, international journal, publishing a paper, call for paper 2012, journal of pharmacy, how to get a research paper published, publishing a paper, publishing of journal, research and review articles, Pharmacy journal, International Journal of Pharmacy, hard copy of journal, hard copy of certificates, online Submission, where to publish research paper, journal publishing, international journal, publishing a paper
Objective: To generate preliminary information about of enteroviruses and Enterovirus 71 (EV71) in patients with aseptic meningitis in Khartoum State, Sudan.
Method: Cerebrospinal fluid specimens were collected from 89 aseptic meningitis patients from different Khartoum Hospitals
(Mohammed Alamin Hamid Hospital, Soba Teaching Hospital, Omdurman Military Hospital, Alban Gadeed Teaching Hospital and Police Hospital) within February to May 2015. Among these 89 patients, 43 (48%) were males and 46 (52%) were females. The patient’s age ranged between 1 day and 30 years old. The collected specimens were assayed to detect enteroviruses and EV71 RNA using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique
Enterocin 55 produced by non rabbit-derived strain Enterococcus faecium EF55 ...Agriculture Journal IJOEAR
— Ent55 is produced by poultry strain Enterococcus faecium EF55. It is substance which can be allotted to Class II enterocins; thermo-stable, small peptide. Because producer strain has shown beneficial effect in poultry and broiler rabbits as well, we decided to apply Ent55 in broiler rabbit husbandry. Ent55 showed antimicrobial activity in broiler rabbits by reduction of staphylococci, Clostridiae, pseudomonads and coliforms. Its beneficial effect was demonstrated by stimulation of phagocytic activity as well as by reduction of Eimeria spp. oocysts. GPx values were lower; it means, no oxidative stress was evoked. Moreover, it has not negative influence on growth performance and biochemical parameters. Our results indicated that enterocin produced by not-autochtonous strain can also have protective and beneficial effect in broiler rabbits.
Protective Effect of Egyptian Propolis Against Rabbit PasteurellosisBee Healthy Farms
Propolis is known for its protective effects on humans and animals, including improving respiratory conditions. It's also documented to be a very complementary adjuvant with other treatment modalities.
Pasteurella multocida is a well known cause of morbidity and mortality in rabbits. The predominant syndrome is upper respiratory disease or "snuffles." P. multocida is often endemic in rabbit colonies and the acquisition of infection in young rabbits is correlated to the prevalence in adult rabbits.
Mobile phone has been source of microorganisms that cause diseases of public health concerns. In a study, one-fi fth of cellular phones examined were found to harbor pathogenic bacteria indicating that these devices may serve as vehicles of transmission. Swab samples were collected aseptically from the phones of different handlers like motor bike riders, food vendors, meat sellers and nursing mothers. Bacteria isolation and identifi cation were carried out using pour plating technique with distinctive morphological and biochemical characteristics.The pathogenicity of the bacterial isolates was investigated through oral inoculation into albino rats. Eighty-eight (88) bacteria were isolated and selected based on their resistance to antibiotics for pathological study. Loss in weight was observed in some albino rat. Along with reduction in the packed cell volume, hemoglobin but raised white blood cell. Animal inoculated with Bacillus cereus showed meningitis like symptom after the first week of inoculation. Also, there were short and stunted villi; low crystal depth with necrotic
debris in the lumen. It has been observed that cell phones may harbor pathogenic bacteria and can subsequently plays role as fomite in the disease transmission. Therefore, the need to educate community phone handlers in the rural area becomes imperative.
ABSTRACT- A number of 18 adults male outbred albino rats, weighing between 133-137g were used to investigate the drug susceptibility of Trypanosoma evansi strain isolated from naturally infected dromedary camels in Umbadir area, North Kordofan State, Sudan. The rats were divided into 3 groups (C, D and F) of 6 animals each. Group C and D were infected intraperitoneally with T. evansi (Umbadir stabilate) with 1×104 Trypanosome for the inoculum. Group D rats were given quinapyramine sulphate (20 mg/Kg bwt) after parasitaemia was evident. Group F was left as healthy uninfected control for the stabilate. When parasite counts were one or more parasites per field, counting in haemocytometer were used for exact number of parasite per cubic millimeter using Neubaeur’s counter. Parasites from tail blood were first fixed, stained and diluted in trypanosome diluting reagent. The parasites were diluted to the level that can be easily counted in WBC counting chamber in the haemocytometer. The total number of parasites was expressed as log10 number of parasites per ml of blood. The presence and degree of parasitaemia were determined daily for each rat by examining tail blood. The identity of the local stabilates of Trypanosoma evansi was confirmed through adopting PCR where primers that target the internal transcribed spacer one (ITS1) of the ribosomal DNA were used. There was significant reduction in serum glucose and potassium as well as significant increase in total protein, urea, calcium, albumin and cholesterol in group C. The Umbadir stabilate showed low mortality and high sensitivity to quinapyramine sulphate.
Key-words- Drug susceptibility, T. evansi, Dromedary camels, Sudan
Hepato Protective Assessment of Pawpaw Leaves, Neem, Lemon Grass and Acts on ...ijtsrd
Malaria is a major concern in Nigeria, and stands as the second leading cause of death from all infectious disease in Africa. Several studies have reported the damaging effect of the parasite to various body organs especially the liver. Reports over time has shown the benefits of various plants extracts in ethno medicine. However, not much have been done on the effects of some of these extracts in combined form on its hepato protective assessment in comparison with any known ACT based anti malaria. The focus of this study was to explore the hepato protective properties of ethanoic extract of Carica papaya Linn, AzadirachtaIndica, CymbopogonCitratusagainst ACT based antimalarial therapy on plasmodium berghei parasitized wistar rats. Phytochemical analysis of the extracts were done according to the method described by Treaseand Evans. Hepato protective assessment were done using the liver function tests and assay of the liver histology respectively. One hundred and ten 110 rats distributed into 11 groups, each group having 10rats were used for the experiment. Negative control received just feed and water, Positive control were induced with the malaria parasite and given feed and water only. The tests groups were induced with malaria, received feed and water and treated with 500mg kg, 250mg kg and 165mg kg doses of the extracts, both individually and in combined forms, as well as the standard ACT anti malaria. Phytochemical screening showed that the plant extracts possessed high concentration of Tannins, Flavonoids, Saponins and Alkaloids. Plasmodium berghei increased the activities of ALP, ASP and ALT when compared with the positive control group. This may be attributed to increase in functional capacity of the liver as a result of the presence of the infection for the tests groups. Treatment with the plant extracts decreased ALP and ALT levels significantly P 0.05 , as well as AST levels except for the Neem extract. Histological examination of the liver of test animals showed no extensive damage to the tissue by the individual extracts when compared to the negative control group. Nnyaha Anthonia E. | Igbokwe Ugochukwu V. | Okonkwo Onyeka Chukwudi | Ajeka Prisca O. | Nwaissac Ikechukwu S. | Okpa Precious N. "Hepato-Protective Assessment of Pawpaw Leaves, Neem, Lemon Grass and Acts on Plasmodium Berghei Parasitized Wistar Rats" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-5 , August 2021, URL: https://www.ijtsrd.com/papers/ijtsrd46292.pdf Paper URL: https://www.ijtsrd.com/medicine/physiology/46292/hepatoprotective-assessment-of-pawpaw-leaves-neem-lemon-grass-and-acts-on-plasmodium-berghei-parasitized-wistar-rats/nnyaha-anthonia-e
Clinical Manifestations of Plasmodium bergheiANKA Infection in Juvenile Mice:...AI Publications
Malaria is an important health and development challenge in Africa, Animalmodels most particularly mice, have long been employedto study malaria pathogenesis. Clinical manifestations due to Plasmodium bergheiANKA infection in juvenile mice as a model for understanding the complications ofcongenital malaria in neonates.Forty-five juvenile mice (5-7 days old) were acquired from University College Hospital, Ibadan and injected with 2 x 107 (0.2ml) Plasmodium berghei ANKA parasitized red blood cells (PRBCs). Mice were transported to the study site, kept in well ventilated cages and fed daily with a balanced ration. Every day after post-P. berghei infection, mice were monitored for mortality. Clinical manifestations ofexperimental cerebral malaria (ECM) was assessed and confirmed if at leastruffled fur, hunching, wobbly gait, limb paralysis, convulsions, or coma was observed. Each sign was given a score of 1. Animals with scores ≥4 were considered to have severe ECM.20 (44%) micewerelost due to natural cause (i.e. stress) at day 2 of the experiment. Between day 4 and 9, 25 (56%) of the studymice presented clinical signs of ECM which includes; ruffled fur 25(100%), hunching 21 (84%), wobbly gait 17 (68%), limb paralysis 20 (80%), convulsions 25 (100%) and subsequently died. Survival rate and severity of ECM in the mice differs, 22 (88.0%) had severe ECM and 3(12.0%) had mild ECM.This study has shown that parasite establishment and malaria complications can manifest as early as 4 days’postP. berghei infection in 5-7 days old mice.
Johne's disease (JD) or Paratuberculosis (PTB) has gained a great attention by many industrial countries for its severing economic losses and possibly zoonotic concerns. In the current study conventional clinical and direct microscopic examination compared to real time polymerase chain reaction (RT-PCR) were used to diagnose JD in clinically suspected small ruminants. Clinical examination revealed 130 (8.7%) suspected cases that showed history of emaciation and diarrhea out of the total examined (1500) animals. Direct microscopy of Ziehl-Neelsen (ZN) stained smears (130) revealed 62 (47.7%) acid fast bacteria resembled Mycobacterium avium subsp. paratuberculosis (MAP). RT-PCR insertion sequence gene (IS900) detected MAP in 25 (65.8%) out of 38 fecal samples harbored acid fast bacilli. We concluded and recommended that RT-PCR considers the most rapid confirmatory method for screening and diagnosis of the MAP in comparison to low specific conventional phenotypic methods, which still remained valuable techniques in the diagnosis of JD in developing countries.
Key-words: Johne's disease, Paratuberculosis, Acid fast bacteria, Ziehl-Neelsen stain, IS900 gene
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research paper publishing, where to publish research paper, journal publishing, how to publish research paper, Call for research paper, international journal, publishing a paper, call for paper 2012, journal of pharmacy, how to get a research paper published, publishing a paper, publishing of journal, research and review articles, Pharmacy journal, International Journal of Pharmacy, hard copy of journal, hard copy of certificates, online Submission, where to publish research paper, journal publishing, international journal, publishing a paper
Objective: To generate preliminary information about of enteroviruses and Enterovirus 71 (EV71) in patients with aseptic meningitis in Khartoum State, Sudan.
Method: Cerebrospinal fluid specimens were collected from 89 aseptic meningitis patients from different Khartoum Hospitals
(Mohammed Alamin Hamid Hospital, Soba Teaching Hospital, Omdurman Military Hospital, Alban Gadeed Teaching Hospital and Police Hospital) within February to May 2015. Among these 89 patients, 43 (48%) were males and 46 (52%) were females. The patient’s age ranged between 1 day and 30 years old. The collected specimens were assayed to detect enteroviruses and EV71 RNA using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique
ABSTRACT- Enteric fever is a major public health problem in developing countries like India. An early and accurate diagnosis is necessary for a
prompt and effective treatment. We have evaluated the diagnostic accuracy of ENTEROSCREEN-WBTM as compared to Widal test in rapid and early
diagnosis of enteric fever. A total of 145 patients serum samples were tested by Rapid ENTEROSCREEN-WBTM and Widal test including clinically
suspected cases of enteric fever of all age groups. Vaccinated individuals, patients on antibiotic therapy, patients who have other associated conditions,
patients suffering from fever due to non-enteric etiology & non consent patients were excluded. The overall sensitivity, specificity, positive
predictive value (PPV) and negative predictive value (NPV) of ENTEROSCREEN-WBTM considering Widal test as gold standard were 50% and
96%, 66.66% and 92.30% respectively. ENTEROSCREEN-WBTM was found to be significantly more specific. Although the Rapid ENTEROSCREEN-
WBTM tests are meant to diagnose of S. typhi. Ten patients who were ENTEROSCREEN-WBTM positive for S. typhi were also positive by
Widal test.
Key words- Enteric fever, Rapid ENTEROSCREEN-WBTM, Non-enteric etiology, S. typhi, Widal test
GeneXpert MTB/RIF: A Useful Tool for Rapid and Accurate Diagnosis of Tubercul...komalicarol
The primary objective of this study was to show the usefulness and
importance of GeneXpert MTB/RIF, a rapid test that simultaneously detects Mycobacterium tuberculosis complex (MTBC) and
resistance to rifampicin (RIF) in less than 2 hours.
1ScIeNtIFIc REPORTS | (2018) 8:1250 | DOI:10.1038/s41598-018-19638-x
www.nature.com/scientificreports
Histology, immunohistochemistry,
and in situ hybridization reveal
overlooked Ebola virus target
tissues in the Ebola virus disease
guinea pig model
Timothy K. Cooper1, Louis Huzella 1, Joshua C. Johnson 1, Oscar Rojas1, Sri Yellayi1,3,
Mei G. Sun2, Sina Bavari2, Amanda Bonilla1, Randy Hart1, Peter B. Jahrling 1, Jens H. Kuhn 1
& Xiankun Zeng 2
Survivors of Ebola virus infection may become subclinically infected, but whether animal models
recapitulate this complication is unclear. Using histology in combination with immunohistochemistry
and in situ hybridization in a retrospective review of a guinea pig confirmation-of-virulence study, we
demonstrate for the first time Ebola virus infection in hepatic oval cells, the endocardium and stroma of
the atrioventricular valves and chordae tendinae, satellite cells of peripheral ganglia, neurofibroblasts
and Schwann cells of peripheral nerves and ganglia, smooth muscle cells of the uterine myometrium
and vaginal wall, acini of the parotid salivary glands, thyroid follicular cells, adrenal medullary cells,
pancreatic islet cells, endometrial glandular and surface epithelium, and the epithelium of the vagina,
penis and, prepuce. These findings indicate that standard animal models for Ebola virus disease are not
as well-described as previously thought and may serve as a stepping stone for future identification of
potential sites of virus persistence.
Ebola virus disease (EVD) is a severe and frequently lethal affliction of humans caused by infection with any
of three members of the mononegavirus family Filoviridae: Bundibugyo virus (BDBV), Ebola virus (EBOV),
and Sudan virus (SUDV). A fourth virus, Taï Forest virus (TAFV), has thus far caused only a single reported
human infection, which was nonlethal1. EVD is an exotic disease with case numbers rarely surpassing the lower
hundreds1; however, from 2013–2016, EBOV caused an EVD outbreak in Western Africa encompassing 28,616
infections and 11,310 deaths in Guinea, Liberia, and Sierra Leone2. Long term sequelae in individual survivors of
acute EVD and the similar Marburg virus disease (MVD) and filovirus persistence followed by disease relapse or
sexual transmission had been reported before this outbreak3–8. However, observations during and following the
Western African EVD outbreak suggest that sequelae and filovirus persistence may be common events9. Reported
sequelae include arthralgia, cardiac valvulopathy, parotid gland inflammation, peripheral paresthesia or dyses-
thesia, and gastrointestinal motility disorders10–14. Semen may contain detectable EBOV RNA for more than 500
days following recovery, and EBOV RNA has been detected in breast milk of a subclinically infected mother15,16.
Replicating EBOV has been isolated from the cerebrospinal fluid of an EVD survivor suffering a disease relapse
and from the aqueous hu.
Two main animal pathogenic subspecies of Mycobacterium avium are M. avium avium (Maa) and M. avium paratuberculosis (Map). Their pathogenicity is host-specifi c, Maa causing avian tuberculosis in poultry whereas Map commonly cross-infects to ruminant.Veterinary diagnosis of M. avium infections is microscopic examination of acid-fast bacilli or culture in Löwenstein-Jensen medium,which are time-consuming and low sensitivity. This present study aimed to apply real-time PCR coupled with High-Resolution Melting (HRM) analysis for differential detection of Maa in Thai domestic ducks. Specifi c primer targeting host-expression dependent (hed) region
was designed, PCR product of Maa were amplifi ed from duck’s tissue lesions whereas Map were amplifi ed from cow and deer. HRM real-time PCR was performed and analyzed. Different HRM patterns were showed and melting temperature were analyzed at 83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C for Map. This technique can detect as few as 102 DNA copies and present high specifi city by negative amplifi cation of other pathogenic bacterial species. This technique is sensitive, specifi c, rapid and does not require fl uorescent probes or post-PCR electrophoresis. Our technique is a possible new tool for the detection of Maa and Map infection in tissue specimens.
Two main animal pathogenic subspecies of Mycobacterium avium are M. avium avium (Maa) and M. avium paratuberculosis (Map). Their pathogenicity is host-specific, Maa causing avian tuberculosis in poultry whereas Map commonly cross-infects to ruminant. Veterinary diagnosis of M. avium infections is microscopic examination of acid-fast bacilli or culture in Löwenstein-Jensen medium,which are time-consuming and low sensitivity. This present study aimed to apply real-time PCR coupled with High-Resolution Melting (HRM) analysis for differential detection of Maa in Thai domestic ducks. Specific primer targeting host-expression dependent (hed) region
was designed, PCR product of Maa were amplified from duck’s tissue lesions whereas Map were amplified from cow and deer. HRM real-time PCR was performed and analyzed. Different HRM patterns were showed and melting temperature were analyzed at 83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C for Map. This technique can detect as few as 102 DNA copies and present high specifi city by negative amplification of other pathogenic bacterial species. This technique is sensitive, specific, rapid and does not require fluorescent probes or
post-PCR electrophoresis. Our technique is a possible new tool for the detection of Maa and Map infection in tissue specimens.
Background
Influenza A viruses are medically significant pathogens responsible for higher mortality and morbidity throughout the world. Swine influenza is known to be caused by influenza A subtypes H1N1, H1N2, and H3N2, which are highly contagious, and belongs to the family Orthomyxoviridae. Efficient and accurate diagnosis of influenza A in individuals is critical for monitoring of a constantly evolving pandemic. A rapid result is important, because timely treatment can reduce disease severity and duration. Rapid antigen tests were among the first-line diagnostic tools for the detection of pandemic H1N1 (2009) virus infection during the initial outbreak. Current study focuses on the significant approach of the usage of molecular method utilizing real-time PCR for the detection of type A influenza virus (H1N1 subtype) in humans.
Methods
A total of 2000 mixed nasal/throat swab specimens collected in commercial viral transport from Apollo hospitals, Hyderabad were submitted to Institute of Preventive Medicine for molecular testing by reverse transcriptase polymerase chain reaction (RT-PCR) from 2009 to 2015 from its affiliated primary care clinics.
Results
Among the 2000 samples collected, 700 samples were positive for Human Inf A, swine Inf A, and Swine Inf H1 (fourth table in the article). One thousand two hundred samples were negative for Human Inf A, swine Inf A, and Swine Inf H1, and 100 samples were positive for Influenza A only.
Conclusion
The molecular testing of H1N1 patients helped the clinicians in timely diagnosis and treatment of these patients during the pandemic surveillance. The RT-PCR test has higher sensitivity and specificity; hence it is considered to be the best tool to use during the pandemic surveillance, as compared to the any other commercial antigen-based tests, which show a variable performance, with the sensitivities of tests from different manufacturers ranging from 9 to 77%.
Seroepidemiology for MERS coronavirus using microneutralisation and pseudopar...Ranawaka A.P.M Perera
We describe a novel spike pseudoparticle neutralisation
assay (ppNT) for seroepidemiological studies on
Middle East respiratory syndrome coronavirus (MERSCoV)
and apply this assay together with conventional
microneutralisation (MN) tests to investigate 1,343
human and 625 animal sera. The sera were collected
in Egypt as a region adjacent to areas where MERS has
been described, and in Hong Kong, China as a control
region. Sera from dromedary camels had a high prevalence
of antibody reactive to MERS-CoV by MERS NT
(93.6%) and MERS ppNT (98.2%) assay. The antibody
titres ranged up to 1,280 and higher in MN assays
and 10,240 and higher in ppNT assays. No other
investigated species had any antibody reactivity to
MERS-CoV. While seropositivity does not exclude the
possibility of infection with a closely related virus, our
data highlight the need to attempt detection of MERSCoV
or related coronaviruses in dromedary camels. The
data show excellent correlation between the conventional
MN assay and the novel ppNT assay. The newly
developed ppNT assay does not require Biosafety Level
3 containment and is thus a relatively high-throughput
assay, well suited for large-scale seroepidemiology
studies which are needed to better understand the
ecology and epidemiology of MERS-CoV.
Applying new techniques to blood assays in cats has enabled researchers to reduce the amount of blood needed nutrition study sample collection studies by 80%, with concomitant benefits for animal welfare. Presented at the Waltham InternationaI Nutrition Science Symposium, October 2016, Chicago
Th1 and Th2 Cytokines Activity during Transformation and Lymphoma Formation S...IOSRJAVS
Marek's disease virus (MDV), a highly transmissible cell-associated neuropathic oncogenic alphaherpes virus affecting poultry health, resulting in considerable economic losses in poultry industry worldwide.Until now MDV still emerging and re emerging causing great economic losses in chicken despite of intensive vaccination and management policy used in poultry farms. However, cytokines and its role in MD pathogenesis and immunity had been described by some workers under certain experimental conditions by using different MDV strains challenge, they need to be more clarified during the more progressive lymphoma transformation stage. The present study aimed to examine the transcriptional profiling of a panel of cytokines genes in the splenic tissues of special broiler Japanese chickens (70-80 days old) contracted natural infection with MDV despite of intensive care and vaccination policy adopted by HVT and CVI988/Rispene. SYBR Green-based, real-time (RT)-PCR protocol was used to quantitate cytokine mRNA in freshly collected spleen tissue of MDV infected and control chicken. Changes in the levels of spleen interleukins (IL) as IL-6, IL-10, IL- 18 and IL-12P35, IL-4, interferon-gamma (IFN-γ) and inducible nitric oxide synthase (iNOS) mRNA was determined. Relative Messenger RNA (mRNA) expression levels of the above mentioned genes, and β-actin as a reference gene, were achieved. The results of quantitative real-time PCR (qPCR) assays using revealed significant up regulation in the expression levels of IL-6, IL-10, IL-18, and IL-12P35). The changes in the mRNA levels of IL-4, IFN-γ and inducible nitric oxide synthesase (iNOS) were minimal and not significant in comparison to those in uninfected age-matched control chicken. In conclusion, these data strongly support the hypothesis that pro-inflammatory responses, including high levels of Th2 cytokines as IL-10 and other interleukins as IL-6, IL-18 and IL12-P35, may play a major role during MDV lymphoma transformation stage induced by MDV strain of high virulence. These cytokines may be involved in maintenance of MDV infection and lymphoma formation. On the other hand, Th1 cytokines as IFN-γ, and iNOS had no or minimal role in induction of MDV-specific immune response during MDV lymphoma transformation stage.
NRAMP1 and VDR Gene Polymorphisms in Susceptibility to Tuberculosis in Venezu...
2004 MHV
1. Immunohistochemical Diagnosis of Mouse Hepatitis
Virus and Mycoplasma pulmonis Infection
with Murine Antiserum
C. T. Liang*,†, S. C. Wu*, Y. T. Huang*, Y. C. Lin*, W. J. Chang*,
J. Y. Chou‡, S. C. Liang*,‡ and C. H. Liu†
*National Laboratory Animal Center, National Applied Research Laboratories, Nan-Kang, Taipei 115, †
Department and
Graduate Institute of Veterinary Medicine, College of Bioresources and Agriculture, National Taiwan University,
Taipei 106, and ‡
Laboratory Animal Center, National Defense Medical Center, Taipei, Taiwan, ROC
Summary
This study established a modified alkaline phosphatase-labelled avidin-biotin-complex (ABC-AP) method
for diagnosis of mouse hepatitis virus (MHV) and Mycoplasma pulmonis infection from formalin-fixed,
paraffin wax-embedded sections, murine antibody-positive serum being used as the primary reagent. With
this method, MHV antigen in cAnNCrj.Cg-Foxn1nu
/Foxn1nu
mice and M. pulmonis antigen in Wistar rats
were immunolabelled in tissue sections. MHV antigen was clearly detected in samples of liver, stomach,
caecal and colonic mucosa, and spleen. M. pulmonis antigen was demonstrated on the luminal surface of
bronchiolar epithelial cells. This method may prove useful in diagnosis when commercial antisera are
unavailable or when immunosuppression prevents serological diagnosis.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: bacterial infection; mouse; mouse hepatitis virus; Mycoplasma pulmonis; viral infection
Introduction
Avidin-biotin-complex immunohistochemistry
offers a sensitive, reliable method for the detection
of pathogens in tissues. Mouse hepatitis virus
(MHV) and Mycoplasma pulmonis are the most
prevalent pathogens of laboratory mice and rats
(National Research Council, 1991). Respiratory
strains of MHV infect the nasal mucosa and then
spread to the liver, lymphoid tissue, uterus,
placenta, peritoneum, brain, vascular endothelium
and bone marrow by the lymphatic or vascular
route, or directly via olfactory pathways from the
nose to the brain. Enterotropic strains of MHV
usually infect the intestinal mucosal epithelium
and nasal passages, with less involvement of
other tissues (National Research Council, 1991;
Compton et al., 1993; Liang et al., 1995).
M. pulmonis, an extracellular pathogen of mice
and rats, preferentially colonizes the luminal sur-
face of respiratory epithelium, the middle ear and
endometrium (National Research Council, 1991;
Percy and Barthold, 1993). Infection is usually
diagnosed by microbial isolation, serological test-
ing and histological examination. Microbial iso-
lation requires multisite culture for reliable results,
and serological methods are hampered by cross-
reactivity between different species of mycoplasma
(Cassell et al., 1981). Serological testing is useful
during the active and convalescent phases of
disease (Feldman, 2001), but screening of immu-
nodeficient mice is unreliable (Casebolt et al.,
1997). Immunohistochemistry is widely used to
J. Comp. Path. 2004, Vol. 131, 214–220
www.elsevier.com/locate/jcpa
0021–9975/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi: 10.1016/j.jcpa.2004.04.003
Correspondence to: C. H. Liu, Department of Veterinary Medicine,
National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei
106, Taiwan.
2. detect microbial antigen in tissue sections from
naturally infected animals, except when specific
primary antibodies are commercially unavailable or
excessively expensive. In such cases, the use of
positive antiserum from infected members of the
homologous species is of potential valve. Although
the use of homologous primary antiserum is not
usually applicable in immunohistochemistry, a
suitable method was recently described by Lu and
Partridge (1998). In the present study, an avidin-
biotin-complex and alkaline phosphatase (ABC-AP)
method was developed in which ELISA-positive
murine antiserum was used as the primary antibody
for the diagnosis of MHVand M. pulmonis infection.
Materials and Methods
Animals
Experiment 1. Specific-pathogen-free cAnNCrj.Cg-
Foxn1nu
/Foxn1nu
and Foxn1nu/þ
nude mice, orig-
inally obtained from Charles River Laboratories
(Yokohama, Kanagawa, Japan), were maintained at
the National Laboratory Animal Center (NLAC) in
Taiwan. Thirty of these mice, consisting of 20
homozygous males aged 5 weeks and 10 homo-
zygous females aged 9 weeks were sold, to a unit
that maintains animals for use in research in Taipei
City, on February 2 and March 8, 2000, respectively.
Animals were housed in sterile microisolator cages
(Laboratory Products, Maywood, NJ, USA), kept in
animal cabinets (Nu-air; Plymouth, MN, USA), and
placed in conventional rooms. Sterile water and
commercial rodent chow (PMI Feeds, St Louis,
MO, USA) was provided ad libitum. Bedding
changes, water replenishment and supply of food
were carried out in a class-II biological safety
cabinet. The animal houses were maintained at
20 to 25 8C with a 12-h light/dark cycle.
At 42–49 days after arrival at the unit, the male
nude mice had become emaciated, anorexic and
dehydrated, with scaly skin, hunched posture,
diarrhoea and ocular discharge, and 50% had
died. The female nude mice showed similar but less
severe signs. Four male and two female nude mice
were humanely killed at the age of 15–16 weeks
and samples were taken back to the NLAC for
pathological examination.
Experiment 2. Sixty 6-week-old female Wistar rats
from the NLAC were transferred to a clean
conventional local area and housed in autoclaved
microisolators. Bedding was changed twice a week
in class II biohazard cabinets. Animals were main-
tained at a room temperature of 20–25 8C and
humidity of 50–70%, with a 12-h light /dark cycle.
Sterile water and commercial rodent chow (PMI
Feeds) were provided ad libitum. Bedding (Beta-
chip; Northeastern Inc., Warrensburg, NY, USA),
polysulfone cages (Laboratory Products) and
supplies were all autoclaved. A proportion (30%)
of the animals showed sneezing 5–6 weeks after
arrival. Thirteen of the affected animals, now aged
12–24 weeks, were killed for diagnostic evaluation.
Necropsy revealed consolidation of the apical and
cardiac lobes of the lungs. ELISA screening for
pneumonia virus of mice (PVM), Sendai virus,
lymphocytic choriomeningitis virus (LCMV), and
sialodacryoadenitis virus (SDAV) was negative. Anti-
body titres of three rats for M. pulmonis, assessed by
ELISA score, were 6.07 to 17.22. ELISA results were
interpreted on the basis of the method described
below.
Preparation of ELISA-positive Sera
The ELISA monitoring programme and diagnostic
service of the NLAC were used to detect the
following infectious agents: pneumonia virus of
mice (PVM), reovirus (Reo-3), Sendai virus, lym-
phocytic choriomeningitis virus (LCMV), Theiler’s
murine encephalomyelitis virus (GD VII), minute
virus of mice (MVM), mouse hepatitis virus (MHV),
mouse adenovirus (Mad), ectromelia virus,
Kilham’s rat virus (KRV), sialodacryoadenitis virus
(SDAV/RCV), M. pulmonis, hantavirus, K virus, and
Clostridium piliforme. The procedure followed the
Charles River ELISA scoring system (Serology
Method Manual; Charles River Laboratories,
Wilmington, MA, USA). Briefly, 50 ml of serum
sample, diluted 1 in 60 in BLOTTO (Bovine Lacto
Transfer Technique Optimizer; 5% non-fat dry milk
in phosphate-buffered saline (PBS)) (Johnson et al.,
1984), were added to each appropriate antigen well
and control well. The plate was covered and
incubated for 40 min at 37 8C. After several wash-
ings, 50 ml of horseradish peroxidase-conjugated,
affinity-purified horse anti-mouse or anti-rat IgG
(Kirkegaard and Perry Laboratories, Maryland,
USA), depending on species, were added to each
well. After incubation for 40 min at 37 8C, the plate
was washed again. One hundred ml of 0.4 mM
ABTS-2.0 mM H2O2 chromogenic substrate were
then added to each well and the plate was
incubated at room temperature for 40 min. Absor-
bance was determined colorimetrically at 405 nm
with an ELISA microplate reader (Thermo-max;
Molecular Devices, Sunnyvale, CA, USA). Absor-
bance values were transmitted from the ELISA
reader to a personal computer (PC) where
they were converted to scores by dividing by 0.13.
Diagnosis of Laboratory Animal Infections 215
3. The denominator of 0.13 divided net absorbance
values of 0.13 to 1.3 into scores of 1 to 10. Integer
scores were read and interpreted by comparison
with the 3-decimal absorbance values. The PC was
also used to compute net scores (Scoreantigen minus
Scoretissue control). A result was considered
non-specific and recorded as tissue control (TC)
when both Scoreantigen and Scoretissue control were 2
or above. Provided that the Score tissue control was
lower than 2 (absorbance values lower than 0.26),
net scores were interpreted as follows: 0–1, nega-
tive; 2–3, borderline; $3, positive. When the test
serum was interpreted as “single agent (i.e., MHV)-
positive”, and the net score was $10, serum samples
were collected and stored at 220 8C until used as
primary antibody for immunohistochemistry.
Pathological Examination
The lungs, trachea, lymph nodes, heart, liver,
spleen, small intestine, stomach, kidneys, urinary
bladder, adrenal glands, skin and brain from
affected animals in experiments 1 and 2 were
fixed in 10% neutral buffered formalin. The tissue
samples were processed by routine methods to
paraffin wax-embedded blocks. Sections (6 mm)
were stained with haematoxylin and eosin (HE).
Immunohistochemistry
The Mouse on Mouse kit (M.O.M.e; Vector
Laboratories, Burlingame, CA, USA) was used for
the immunohistochemistry study of MHV infection
in experiment 1. The primary antibodies were
ELISA-positive, mouse anti-MHV sera (ELISA
score; 11.2–14.4). Infection with PVM, Reo-3,
Sendai virus, LCMV, GD VII, MVM, Mad, ectrome-
lia virus, K virus, M. pulmonis and polyoma virus
were ruled out on the basis of ELISA results.
Tissue sections were dewaxed in xylene and
rehydrated in a graded alcohol series. Antigen
unmasking was performed by immersion of sec-
tions in Vector antigen unmasking solution 1% in
PBS and boiling for 5 min in a 1450-W microwave
oven (RE-C102; Sampo Co., Taiwan). The sections
were then immersed in cool PBS for 10 min, rinsed
in PBS, and incubated with trypsin (Sigma Chemi-
cal Co., St Louis, MO, USA) 0.1% in PBS for 5 min
at 40 8C. Endogenous peroxidase activity was
quenched with hydrogen peroxide 0.3% in metha-
nol for 5 min at 40 8C. The sections were then
rinsed in PBS, incubated for 30 min at 40 8C in
mouse M.O.M.e IgG blocking reagent, rinsed with
PBS, and incubated in M.O.M.e diluent for 5 min
at 40 8C. Subsequently, they were incubated in
ELISA-positive murine anti-MHV serum (diluted 1
in 60 in M.O.M.e diluent) as the primary antibody
for 24 h at 4 8C. Substitution of PBS or mouse
serum (negative for any pathogen) for the primary
antibody served as a negative control. The sections
were then rinsed in PBS, incubated with M.O.M.e
biotinylated anti-mouse IgG reagent for 60 min
at 40 8C, rinsed in PBS, incubated in Vectastainw
ABC-AP reagent for 60 min at room temperature,
rinsed in PBS, and incubated with alkaline phos-
phatase substrate solution (Vectorw
Red; Vector
Laboratories) in 100 mM Tris HCl, pH 8.2–8.5, for
30 min at room temperature. Endogenous alkaline
phosphatase activity of tissues was inhibited
by adding one drop of levamisole (Vector
Laboratories) to 5 ml of Tris HCl buffer before
preparation of the substrate working solution. The
sections were rinsed in distilled water, counter-
stained with Mayer’s haematoxylin and examined
microscopically.
Lung sections from experiment 2 were pre-
treated as described in experiment 1. They were
then rinsed in PBS, incubated for 30 min at 40 8C in
diluted (1 in 20) normal horse serum (Vector
Laboratories), incubated with diluted (1 in 60)
ELISA-positive murine anti-M. pulmonis serum
(ELISA score:13.4–17.7) as primary antibody for
24 h at 4 8C, rinsed in PBS, and incubated with
diluted biotinylated horse anti-mouse IgG second-
ary antibody (1 in 100, rat-absorbed) for 60 min at
40 8C. The subsequent procedures were the same as
those in experiment 1.
Results
ELISA Monitoring Results
In the MHV test, 485 mouse serum samples were
examined. Of these, 465 were negative (score ,3),
three were positive (score 3–10), and 17 had a high
MHV score (.10). Seven of 17 samples with a high
score were excluded because of simultaneous
positivity for GDVII. Ten serum samples (ELISA
score: 11.2–14.4) were collected for further diag-
nosis of MHV infection in experiment 1.
In the M. pulmonis test, 268 rat serum samples
and 383 mouse serum samples were examined.
Only six rat samples were positive, and 380 mouse
samples were negative (score ,3). Three mouse
samples showed a high M. pulmonis score (.10),
but one of these was excluded because of simul-
taneous positivity for MHV. The remaining two
mouse serum samples (ELISA score: 13.4–17.7)
were used for the diagnosis of M. pulmonis infection
in Wistar rats in experiment 2. These Wistar rats
C.T. Liang et al.216
4. were confirmed as having M. pulmonis infection by
testing 13 rat serum samples; three rats (23%) had
an ELISA score of 6.07–17.22, and four (31%) had
typical pulmonary lesions.
Histopathology and Immunohistochemistry
Experiment 1. Gross examination of the four affected
male nude mice revealed that the liver was firm and
pale with multiple, white, depressed foci, 2 to 3 mm
in diameter. Microscopically, necrotic foci were
seen to be scattered throughout the hepatic
parenchyma, being well demarcated from the
adjacent normal tissues (Fig. 1). Multinucleated
syncytial giant cells with basophilic cytoplasmic
granules (20 mm in diameter) were often present.
Colonic and caecal mucosal ridges were attenuated
and shortened, and contained syncytial cells
(Fig. 2). Immunohistochemical results revealed
MHV antigen at the periphery of the necrotic foci
in the liver (Fig. 3) and within the caecal and
colonic multinucleated syncytial cells (Fig. 4),
spleen, and jejunal and gastric mucosa.
Experiment 2. Microscopical examination of two
affected Wistar rats showed extensive consolidation
of the lung, with variable degrees of hyperplasia
and metaplasia of bronchiolar epithelial cells, and
mononuclear cell infiltration into the bronchioles
and adjacent alveolar spaces. Loss of cilia and
flattening of epithelial cells in the bronchioles were
noted (Fig. 5). Aggregates of necrotic debris and
neutrophils were present in the bronchiolar
lumina. Peribronchial and perivascular cuffing
by lymphocytes, macrophages and plasma cells
was also observed. Sections of lung showed
immunohistochemical labelling for M. pulmonis
Fig. 1. MHV-infected mouse liver showing necrosis with multi-
nucleated syncytial giant cells (arrow) at the periphery
of the lesion. HE. Bar, 50 mm.
Fig. 2. Syncytial cells (arrowhead) in the surface epithelium of
the colon of a MHV-infected mouse. HE. Bar, 25 mm.
Fig. 3. MHV antigen within necrotic hepatocytes and syncytial
cells (arrowhead) immunolabelled with ELISA-positive
serum as primary antibody. ABC-AP with haematoxylin
counterstain. Bar, 25 mm.
Fig. 4. Colonic epithelial and syncytial cells (arrowheads)
immunolabelled for MHV with ELISA-positive sera.
ABC-AP with haematoxylin counterstain. Bar, 25 mm.
Diagnosis of Laboratory Animal Infections 217
5. antigen over the luminal surface of hyperplastic
bronchiolar epithelial cells (Fig. 6).
Discussion
Only 10 serum samples with a positive ELISA titre
for MHV alone and two with a positive titre for
M. pulmonis alone were used in this study. The
results indicated the applicability of the technique
to diagnosis in the absence of access to commer-
cially produced antibodies. There have been few
reports of the use of ELISA-positive murine sera as
primary antibody for the immunohistochemical
examination of rodent tissue. Polyclonal antiserum
has been used for the diagnosis of MHV and
M. pulmonis infection (Brownstein and Barthold,
1982; Brunnert et al., 1994; Liang et al., 1995). MHV
is the most common viral pathogen of mice, and
seropositivity for MHV had been reported in
19–83% of animals in mouse colonies (Kraft and
Meyer, 1986, 1990; Casebolt et al., 1988; National
Research Council, 1991). In Taiwan, MHV is highly
prevalent in mouse colonies, especially in immu-
nosuppressed mice (Liang et al., 1995). Due to the
inability of immunodeficient homozygous mice to
produce antibody, immunohistochemistry may
represent a useful replacement for serology in
diagnosing MHV infection. In this study, high-titre
serum from immunocompetent mice was used as
primary antibody. The findings regarding the
distribution of MHV antigen in experiment 1
accorded with those of previous reports (Weir
et al., 1987; Barthold et al., 1990), the antigen
being demonstrated in the intestine, liver, spleen
and stomach, consistent with multi-organ MHV
infection (Compton et al., 1993).
Serology and culture are widely used in the
diagnosis of M. pulmonis infection, but discrepan-
cies sometimes occur (Cassell et al., 1981). ELISA
has detected M. pulmonis infection in 8–78% of rat
colonies and 35–91% of mouse colonies, depend-
ing on whether conventional or barrier-maintained
facilities are used (Casebolt et al., 1988; Kraft and
Meyer, 1990). An advantage of ELISA is the low
incidence of non-specific or false positive reactions
as compared with haemagglutination inhibition
(HI) (Kraft and Meyer, 1986). Discrepant results
for M. pulmonis infection obtained by different
serological tests may be due to reactive substances
in the serum, such as lysozyme, antinuclear
antibodies, protease and bacterial products
(LaRegina et al., 1987). Culture of M. pulmonis
from tracheobronchial lavage fluid showed 89.6%
positivity in rats and 36.5% positivity in mice in
non-barrier-maintained facilities (Timenetsky and
DeLuca, 1998). For routine monitoring of
M. pulmonis, the preferred use of time-consuming
culture procedures as opposed to serological
testing is applicable only in acute or early infection.
One-third of infected animals do not yield
M. pulmonis in culture (Kraft et al., 1982). Culture
and histopathology may be misleading in evaluat-
ing a colony of rodents for mycoplasma infection,
particularly when the prevalence is low (Cox et al.,
1988).
M. pulmonis infection in the chronic stage is
readily detected histopathologically (Kraft et al.,
1982; Goto et al., 1994), but in some instances MHV
or mycoplasmal infection produces minimal or no
lesions. In such instances, immunohistochemistry
is valuable (Matthaei et al., 1998). In experiment 2,
labelling of M. pulmonis antigen was noted on
Fig. 5. Hyperplasia of bronchiolar epithelial cells of a rat
infected with M. pulmonis. HE. Bar, 25 mm.
Fig. 6. M. pulmonis antigen is clearly demonstrated on the
luminal surface of hyperplastic bronchiolar epithelial
cells. ABC-AP with haematoxylin counterstain. Bar,
25 mm.
C.T. Liang et al.218
6. the luminal surface of bronchiolar epithelial cells, a
site also affected by the cilia-associated respiratory
(CAR) bacillus (Matsushita et al., 1987). In a
previous study (Brunnert et al., 1994) the ABC
method failed to detect M. pulmonis in formalin-
fixed lung tissue but gave 27.4% (17/62) positive
results with ethanol-fixed lung tissue; this com-
pared with 96% (60/62) positive results given by
the polymerase chain reaction (PCR). However,
MHV antigens were demonstrated in formalin-
fixed, paraffin wax-embedded blocks for as long
as 2 years after preparation (Brownstein and
Barthold, 1982). Immunohistochemistry, com-
monly used to detect rodent pathogens, takes
much less time than that required for culturing
mycoplasmas. In the present study, however, the
incubation with primary antiserum was carried out
overnight at 4 8C (Miller and van der Maaten,
1989), rather than for 30 min at room temperature
or at 40 8C (Liu et al., 1997; Liang et al., 2000), the
purpose being to increase the immunolabelling.
PCR is more sensitive than immunohistochem-
istry or microbial isolation but requires a high
degree of technical expertise, and contamination
leads to false positivity (Brunnert et al., 1994). It
offers an advantage over serological testing, how-
ever, in situations in which an antibody response is
unlikely to be generated. In conclusion, the
method described, in which ELISA-positive serum
was used as primary antibody, may be useful in
diagnosing infection in immunodeficient animals
or when commercial immunohistochemical
reagents are unavailable (Matthaei et al., 1998).
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Received; May 6th; 2003
Accepted; April 7th; 2004
C.T. Liang et al.220