J. Seva

Endogenous Interleukin-12 Is Not Required for Resolution of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection in Mice

ABSTRACT A Th1 immune response involving gamma interferon (IFN-γ) production is required to eliminate Chlamydophila abortusinfections. In this study, the role of interleukin-12 (IL-12) in protecting against C. abortus infection was investigated using IL-12−/− and wild-type (WT) C57BL/6 mice to determine the role of this Th1-promoting cytokine. IL-12−/− mice were able to eliminate the C. abortus infection in a primary infection. However, there was a delay in the clearance of bacteria when IL-12−/− mice were infected with a sublethal dose of C. abortus, the delay being associated with a lower production of IFN-γ. The low level of IFN-γ was essential for survival of IL-12−/−infected mice. Both WT and IL-12−/− mice developed a Th1 immune response against C. abortus infection, since they both produced IFN-γ and immunoglobulin G2a antibody isotype. In addition, when mice were given a secondary infectious challenge withC. abortus, a protective host response which resolved the secondary infection was developed by both WT and IL-12−/−mice. The lack of IL-12 resulted in few infiltrating CD4+ T cells in the liver relative to the number in WT mice, although the number of CD8+ T cells was slightly higher. The more intense Th1 response presented by WT mice may have a pathogenic effect, as the animals showed higher morbidity after the infection. In conclusion, these results suggest that although IL-12 expedites the clearance of C. abortus infection, this cytokine is not essential for the establishment of a protective host response against the infection.

Study of lymphocyte subpopulations in peripheral blood and secondary lymphoid organs in the goat using monoclonal antibodies to surface markers of bovine lymphocytes.

Monoclonal antibodies (mAbs) to surface markers of bovine lymphocytes MHC I, MHC II, B-cells, T-cells (CD2, CD4, CD8 and gamma/delta) and interleukin-2 (IL-2) receptor were tested in the goat by flow cytometry and using immunohistochemical methods. Samples from peripheral blood and secondary lymphoid organs (mesenteric lymph nodes, spleen and ileal Peyers patch) were studied. The percentage of positive cells obtained by flow cytometry and its compartmentalisation in different tissue sections showed that the mAbs against MHC I, MHC II, CD2, CD4, CD8, gamma/delta and IL-2 receptor recognised lymphocyte subpopulations similar to those present in the bovine. However, the mAbs tested on B-cells reacted only partially in the recognition of this subpopulation.

Caprine tuberculosis eradication program: an immunohistochemical study

We studied the evolution of different forms of tuberculosis in herds which are periodically submitted to the comparative tuberculin intradermal reaction (CTID) test within the Caprine Tuberculosis Eradication Program in the Region of Murcia (Spain). In the study, 135 goats with tuberculosis from different herds were diagnosed by histopathological and immunocytochemical techniques. Most animals (58 of the 135) were in the primary complex of tuberculosis, with few or no acid-fast bacilli (AFB) and mycobacterial antigens. Generalized tuberculosis was present in 31 of the 135 animals, and the numbers of bacilli and positive immunocytochemical particles were higher than in animals with the primary complex. Postprimary phase was observed in 44 of the 135 animals, and the number of bacilli and positive immunocytochemical particles increased in proportion to the extent and gravity of the necrotic foci. Tuberculous pneumonia with a high number of bacilli was only observed in two goats. Extrapulmonary tuberculosis was present with lesions in the intestines (60/135), liver (80/135) and spleen (77/135). This study confirmed that the Caprine Tuberculosis Eradication Program is resulting in fewer animals with postprimary tuberculous processes, and a corresponding increase in the number of primary complex and generalized tuberculosis.

Thoracic omentalization for long-term management of neoplastic pleural effusion in a cat.

CASE DESCRIPTION An 11-year-old neutered female domestic longhair cat was evaluated because of a 1-week history of progressive dyspnea, signs of depression, and loss of appetite. A histiocytic sarcoma had been excised from the mammary gland 6 weeks earlier. CLINICAL FINDINGS Physical examination findings were consistent with pleural effusion, and thoracic and abdominal radiography and ultrasonography revealed pleural effusion, a thoracic mass involving the aorta and pulmonary artery, and a caudal abdominal mass that most likely represented enlarged iliac lymph nodes. Cytologic examination of the pleural fluid and fine-needle aspirates from the iliac and right popliteal lymph nodes revealed abundant cells with neoplastic characteristics of indeterminate origin. The clinical diagnosis was generalized malignant neoplasia. TREATMENT AND OUTCOME Pleural drainage was necessary every 5 to 6 days. Exploratory thoracotomy and biopsy of the mass were recommended for better characterization of the thoracic disease. Simultaneously, palliative treatment by advancement of the omentum into the thorax was performed. A final diagnosis of disseminated histiocytic sarcoma was made, and treatment with doxorubicin was begun after surgery. During the 13 months after surgery, the cat was free from signs of respiratory tract disease and had normal activity levels with good exercise tolerance. Fifteen months after surgery, the cats clinical condition worsened and the cat died. CLINICAL RELEVANCE Findings suggested that thoracic omentalization may be considered for palliative treatment of cats with refractory neoplastic pleural effusion when frequent thoracocentesis is necessary and other treatments are not suitable.

Evaluation of the single cervical skin test and interferon gamma responses to detect Mycobacterium bovis infected cattle in a herd co-infected with Mycobacterium avium subsp. paratuberculosis.

This study reports the performance of the single intradermal tuberculin (SIT) test and the interferon-gamma (IFN-γ) assay for Mycobacterium bovis in a cattle herd with high prevalence of paratuberculosis (PTB). A total of 58/350 animals were selected for necropsy based on one or more of the following criteria: positive to SIT, IFN-γ, a breeding cow that seroconverted to PTB and showed signs compatible with a wasting disease. Infection status was determined by post mortem diagnostic tests that included histopathology examination, mycobacterial cultures and PCR identification for M. bovis and Mycobacterium avium subsp. paratuberculosis (MAP). In 7/58 animals primary tuberculosis (TB) lesions, affecting only the retropharyngeal and/or mediastinal lymph nodes, were found; 3/7 animals were found SIT positive. PTB was confirmed in 35/58 animals, of which 30 had seroconverted and 14 had typical clinical signs. 45/58 animals were IFN-γ(+) using the most stringent criterion (cut-off point ≥ 0.05); however, IFN-γ test was only positive in 33 animals when using a higher threshold (cut-off point ≥ 0.1). Three animals co-infected also showed extensive TB and diffuse PTB lesions. These results show that the combined use of SIT and IFN-γ, as interpreted using official guidelines, detected all confirmed cases of TB. Individually, the sensitivity of the SIT was inadequate to diagnose TB-positive animals with an advanced stage of PTB. The large number of IFN-γ(+) animals with no visible TB lesion could be due, in part, to some protection conferred by prior infection with MAP.

Left perinephric abscess associated with nephrolithiasis and bladder calculi in a bitch.

An eight-year-old, entire female Pekingese cross, weighing 3–8 kg, had been inappetent with fever, depression, abdominal pain, vomiting and diarrhoea for seven days. The radiographic and ultrasonographic findings were consistent with glomerulonephritis, nephrolithiasis in both kidneys, bladder calculi and an accumulation of fluid in the left perinephric space. The clinical signs, together with the results of the diagnostic imaging, suggested that this fluid could be pus. A definitive diagnosis of a subcapsular abscess in the left kidney was established when this kidney was removed surgically. A histopathological examination of the kidney revealed a diffuse suppurative interstitial nephritis, membranous glomerulonephritis and an abscess invading the perinephric adipose tissue from the renal cortex. Twelve months after surgery the dog remains clinically stable, but owing to the disease of its remaining kidney its long-term prognosis is poor.

POSTNATAL DEVELOPMENT OF LYMPHOCYTE SUBPOPULATIONS IN THE INTESTINAL MUCOSA IN GOAT

The incidence and location of CD2+, CD4+, CD8+ and gamma/delta T-lymphocytes and B-lymphocytes were studied in the intestinal mucosa in goats of one week, one month, three months and seven months old, using monoclonal antibodies and immunohistochemical methods. At all ages CD2+ lymphocytes were the major subpopulation in the intestinal mucosa, more numerous in the small than in the large intestine and in the villi than in the crypt region. In one week-old animals most of CD2+ lymphocytes were scattered in the lamina propria and the number of lymphocytes that expressed CD4 was less than those that expressed CD8, resulting in a CD4/CD8 ratio less than 1. CD2+ lymphocytes increased markedly until one month old, with a higher increase of CD4+ subpopulation than CD8+ with an inversion of the CD4/CD8 ratio. CD4+ lymphocytes were localised in the lamina propria forming clusters, whereas CD8+ lymphocytes were scattered peripherally under the epithelium and in older animals from three month old, CD8+ lymphocytes were also abundant between epithelial cells. The gamma/delta T-lymphocytes recognised with the mAb CACTB6A represented a minor subpopulation located between epithelial cells or in the lamina propria, no evident changes were observed with age. IgM+ cells were a minor subpopulation in all animals (never higher than 1 cell per 1500 microns-2) located in the crypt region of the lamina propria.

Granulomatous hepatitis caused by Salmonella Typhimurium in a spur-thighed tortoise (Testudo graeca)

and subjected to histopathological examination using haematoxylin and eosin, Gram and Giemsa staining techniques. Macroscopic examination revealed numerous nodular areas in the liver, varying in size up to 0·5 cm in diameter, yellowish and soft to cut (Fig 1). Histopathologically, numerous foci of necrosis were observed in the liver, varying in size from a few necrotic hepatocytes to large areas of necrosis where the structure was partially conserved, surrounded by infiltrations of mononuclear cells (Fig 2). For a microbiological study, samples of affected liver were pre-enriched with a non-selective broth, enriched in tetrathionate broth (Merck) and finally plated on to solid media (agar with xylose, lysine and desoxycholate, and brilliant green agar). The identification of suspicious colonies was performed with the commercial API 20E kit (bioMerieux) and complemented by reactions used as discriminatory biochemical tests, such as beta-galactosidase (ortho-nitrophenyl-beta-D-galactopyranoside), dulcitol, malonate and gelatine hydrolysis (Le Minor 1984). Biochemical identification up to the subspecies level was based on Holt and others (1994). The serovar was determined by serotyping according to the Kauffmann-White scheme (Le Minor 1984, Quinn and others 1994), using slide agglutination tests based on monospecific sera for the 1, 4, 5 and 12 somatic (O) antigens and monospecific flagellar (H) phase 1 (i) and phase 2 (1,2) antigens (Sanofi Diagnostics Pasteur). On the basis of these tests the isolate was identified as S Typhimurium. Infection by Salmonella species in tortoises is frequently caused by serotypes of the S enterica and S arizonae subgroups. To date, the lesions described have been limited to alterations in the intestinal tract, but no clinical cases of hepatic lesions have been reported (Dimow 1966, Pasmans and others 2002). In Spain, a wide variety of serotypes (Abony, Luke, Ohio, Penarth, Poona, Postdam, Typhimurium and others) have been isolated from tortoises (Anon 1991). In avian and reptile species it is considered that there must be a septicaemic process in order to generate lesions in organs other than the gastrointestinal tract such as necrotic foci in the liver (Madewell and McChesney 1975, Greenberg and Sechter 1992, Tellez and others 2002). There is no evidence that the Salmonella serotypes found in tortoises can cause septicaemia (Corsalini 1975, Pasmans and others 2000, 2002b). To date, clinical typhoid hepatitis has not been described in reptiles, but in the present case, the necrotic areas observed in the liver had the same characteristics as the typhoid nodules produced by S Typhimurium in other vertebrates (Baumler and others 1998). Although clinical Salmonella infections in tortoises appear to be relatively rare, in this case lesions were found in the liver of a spur-thighed tortoise that were caused by S Typhimurium; the authors believe that the source of the infection came from the tortoise’s contact with birds, human beings and mice (mouse excreta had been observed in the tortoises’ habitat). Granulomatous hepatitis caused by Salmonella Typhimurium in a spur-thighed tortoise (Testudo graeca)

Acute phase proteins as a tool for differential diagnosis of wasting diseases in growing pigs

The concentrations of haptoglobin (Hp), C–reactive protein (CRP) and serum amyloid A (SAA) were measured in wasted pigs, first to evaluate their usefulness in the diagnosis of infectious, wasting diseases in pigs, and second, to evaluate whether their concentrations can distinguish the lymphoid depletion score in the lymph tissues of wasted affected pigs. Fifty–three wasted pigs and seven specific pathogen free (SPF) pigs were postmortem examined. Gross lesions were evaluated and samples for histopathological, immunohistochemical, molecular biology and microbiological analysis were taken. Thirty–one pigs were diagnosed as postweaning multisystemic wasting syndrome (PMWS) and 22 as porcine respiratory disease complex (PRDC). Lymphoid depletion degree in lymph tissues of PMWS and PRDC affected pigs was determined. Serum Hp was significantly higher in pigs with PRDC in comparison with the PMWS affected pigs. Serum CRP concentration was significantly lower in pigs with PRDC than in PMWS affected pigs (P<0.001). CRP and SAA levels increased with the lymphoid depletion score, presenting statistical differences between pigs with no depletion and pigs with low, moderate or severe lymphoid depletion (P<0.05, P<0.05 and P<0.001 for CRP and P<0.01, P<0.01 and P<0.01 for SAA, respectively). Hp was higher in pigs with no or low depletion compared with the pigs suffering severe lymphoid depletion (P<0.001 and P<0.05, respectively).

Expression of steroid receptors and calponin in a cervical leiomyoma in a young pig.

Thickening of the uterine cervix and bilateral ovarian cystic change was identified in a 6-month-old pig during routine abattoir inspection. Microscopically, the cervical lesion comprised a non-encapsulated mass of densely packed, large and monomorphic spindle cells within the myometrium. Immunohistochemically, the majority of these neoplastic cells expressed the cytoplasmic terminal smooth muscle differentiation marker calponin, the nuclear oestrogen receptor alpha and the progesterone receptor. The ovarian cysts were classified as follicular cysts. A diagnosis of leiomyoma of the uterine cervix with bilateral ovarian follicular cysts was made. The expression of calponin as a marker of smooth muscle differentiation in tumours of the genital tract of the pig has not previously been reported. The expression of steroid hormone receptors suggests a role for steroid hormones derived from the ovarian follicular cysts in tumourigenesis.