Resveratrol modulates Toxoplasma gondii infection induced liver injury by intervening in the HMGB1/TLR4/NF-κB signaling pathway.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that can cause liver diseases in the host, including hepatitis and hepatomegaly. High mobility group box 1 (HMGB1) is the main inflammatory mediator causing cell injury or necrosis. HMGB1 binds to toll like receptor 4 (TLR4), then activates the nuclear factor-κB (NF-κB) signaling pathway, which promotes the release of inflammatory factors. Our previous studies showed that HMGB1 mediated TLR4/NF-κB signaling pathway plays an important role in liver injury induced by T. gondii infection. Resveratrol (RSV) is a small polyphenol, which has anti-inflammatory, anti-cancer, anti-T. gondii effect. However, the effect of RSV on liver injury caused by T. gondii infection is unclear. This study used the RH strain tachyzoites of T. gondii to infect murine liver line, NCTC-1469 cells to establish an in vitro model and acute infection of mice for the in vivo model to explore the protective effect of RSV on liver injury induced by T. gondii infection. The results showed that RSV inhibited the proliferation of T. gondii in the liver, reduced the alanine aminotransferase/aspartate aminotransferase levels and pathological liver damage. Additionally, RSV inhibited the production of tumor necrosis factor-α, inducible nitric oxide synthase and HMGB1 by interfering with the TLR4/NF-κB signaling pathway. These results indicate that RSV can protect liver injury caused by T. gondii infection by intervening in the HMGB1/TLR4/NF-κB signaling pathway. This study will provide a theoretical basis for RSV treatment of T. gondii infection induced liver injury.

Disseminated Neospora caninum infection in a dog with severe colitis.

A 12-y-old spayed female Schipperke dog with a previous diagnosis of inflammatory bowel disease was presented with a 2-mo history of severe colitis. The patient's condition progressed to hepatopathy, pneumonia, and dermatitis following management with prednisolone and dexamethasone sodium phosphate. Colonic biopsies identified severe necrosuppurative colitis with free and intracellular parasitic zoites. Postmortem examination confirmed extensive chronic-active ulcerative colitis, severe acute necrotizing hepatitis and splenitis, interstitial pneumonia, ulcerative dermatitis, myelitis (bone marrow), and mild meningoencephalitis with variable numbers of intracellular and extracellular protozoal zoites. PCR on samples of fresh colon was positive for Neospora caninum. Immunohistochemistry identified N. caninum tachyzoites in sections of colon, and a single tissue cyst in sections of brain. Administration of immunosuppressive drugs may have allowed systemic dissemination of Neospora from the intestinal tract.

Typhlohepatitis and Amyloidosis Associated with High Mortality in Chukar Partridges (Alectoris chukar).

Twelve chukar partridges (Alectoris chukar) from a farm experiencing poor uniformity and increased mortality of up to 65% were submitted for diagnosis. Several birds had mild to moderate multifocal white foci or multifocal petechial hemorrhages throughout the liver. Livers and spleens of older birds were moderate to severely diffusely enlarged. In addition, some birds had caseous cores mixed with blood within the ceca as well as segmentally thickened cecal walls. Histopathology showed acute, multifocal, severe, often coalescing foci of necrosis with accumulation of fibrin and/or fibrinosuppurative inflammation in livers and spleens. Scattered within exudate were protozoa that were spherical or round and measured 12-20 µm in diameter. In the ceca, acute necrosis of the mucosa was observed, often with ulceration and fibrinosuppurative inflammation. Immunohistochemistry using an antiserum against Tritrichomonas foetus revealed round protozoa in ceca, small intestines, liver, spleen, and lung. Quantitative PCR to detect DNA of Histomonas meleagridis was negative. Non-species-specific PCRs amplifying the partial rDNA, the internal transcribed spacer (ITS) region, and the partial beta-tubulin gene yielded products of the expected size. Sequences of the PCR products had the highest homology to sequences of Tetratrichomonas gallinarum and less homology to sequences of H. meleagridis. In addition there was accumulation of amyloid in the space of Disse in the liver, splenic sinuses, and walls of the blood vessels. The typhlohepatitis and other inflammatory processes that were diagnosed might be the underlying cause of the amyloidosis. Other findings were clusters of Clostridium perfringens associated with the lesions in the ceca; multifocal granulomas in the lungs, occasionally associated with fungal hyphae; hyperkeratosis associated with bacteria and Candida sp. cells in the crop; mild infection of the bursal mucosa with Cryptosporidium.

Tiflohepatitis y amiloidosis asociadas con alta mortalidad en perdices chukar (Alectoris chukar). Doce perdices chukar (Alectoris chukar) de una granja con baja uniformidad y alta mortalidad de hasta el 65% se presentaron para diagnóstico. Varias aves presentaron áreas blancas multifocales de leves a moderadas o hemorragias petequiales multifocales en todo el hígado. Los hígados y los bazos de las aves con mayor edad estuvieron agrandados de tamaño de manera difusa y de moderado a severo. Además, algunas aves tenían contenidos caseosos mezclados con sangre dentro de los ciegos, así como paredes cecales engrosadas de manera segmentaria. La histopatología mostró focos de necrosis agudos, multifocales, graves, a menudo coalescentes con acumulación de fibrina y/o inflamación fibrinosupurativa en hígados y bazos. Dispersos dentro del exudado se encontraban protozoarios que eran esféricos o redondos y que medían de 12 a 20 µm de diámetro. En el ciego, se observó necrosis aguda de la mucosa, a menudo con ulceración e inflamación fibrinosupurativa. La inmunohistoquímica con un antisuero contra Tritrichomonas foetus reveló protozoarios redondos en el ciego, intestino delgado, hígado, bazo y pulmón. El método de PCR cuantitativo para detectar el ADN de Histomonas meleagridis fue negativo. Los métodos de PCR no específicos de especie que amplifican parcialmente al rDNA de la región espaciadora transcrita interna (ITS) y el gene parcial de la beta-tubulina dieron productos del tamaño esperado. Las secuencias de los productos de PCR tuvieron la mayor similitud con las secuencias de Tetratrichomonas gallinarum y menos similitud con las secuencias de H. meleagridis. Además, se observó acumulación de amiloide en el espacio de Disse en el hígado, en senos esplénicos y paredes de los vasos sanguíneos. La tiflohepatitis y otros procesos inflamatorios que se diagnosticaron pueden ser la causa subyacente de la amiloidosis. Otros hallazgos incluyeron grupos de Clostridium perfringens asociados con las lesiones en el ciego; granulomas multifocales en los pulmones, ocasionalmente asociados con hifas fúngicas; hiperqueratosis asociada a bacterias y Candida spp. en el buche, e infección leve de la mucosa bursal con Cryptosporidium.

Angiostrongylus vasorum Causing Severe Granulomatous Hepatitis with Concurrent Multiple Acquired PSS.

A 14 mo old female Jack Russell terrier presented with a 12 hr history of vomiting and inappetence. She was subsequently diagnosed with multiple acquired portosystemic shunts during an exploratory celiotomy. Gross and histopathological hepatic abnormalities were consistent with chronic disease, including features suggestive of portal hypertension that was potentially caused by migrating and resident Angiostrongylus vasorum larvae. Fecal analysis and polymerase chain reaction of hepatic tissue confirmed the presence of Angiostrongylus vasorum . The dog recovered clinically following empirical treatment and supportive care. A lack of parasite burden was confirmed 9 wk postdiagnosis; however, serum biochemical analysis at that time was suggestive of ongoing hepatic dysfunction.

Rattus norvegicus como indicador de la circulación de Capillaria hepatica y Taenia taeniaeformis en la Plaza Minorista de Medellín, Colombia / Rattus norvegicus as an indicator of circulation of Capillaria hepatica and Taenia taeniaeformis on a groceries trade center of Medellín, Colombia

Introducción. Rattus norvegicus cumple un papel epidemiológico en el mantenimiento y dispersión de agentes zoonóticos bacterianos, virales y parasitarios de interés en salud pública. La presencia de infección por helmintos en especies Rattus cercanas a poblaciones expuestas en condiciones ambientales propicias, puede convertirse en un factor de riesgo de transmisión. Objetivo. Reportar la frecuencia de infección con Capillaria hepatica y formas larvarias de Taenia taeniaeformis en ratas silvestres (R. norvegicus) capturadas en una zona urbana de Medellín. Materiales y métodos. Se capturaron 254 ejemplares de R. norvegicus. Los hígados de 54 ejemplares que presentaron lesión hepática macroscópica durante la necropsia, fueron examinados por histopatología convencional. Resultados. La frecuencia de infección por C. hepatica fue de 20,1 % (51/254). Seis hígados fueron también positivos para larvas de T. taeniaeformis con una frecuencia de 2,4 % (6/254). Los hígados infestados con C. hepatica exhibían parásitos en el estadio adulto o juvenil y huevos ovalados con opérculos bipolares, asociados con hepatitis granulomatosa leve a moderada multifocal y acompañada por infiltrado leucocitario. Se observaron lesiones granulomatosas en resolución y fibrosis residual o calcificada que contenía huevos. Donde se encontraron cisticercos de T. taeniaeformis, el hallazgo más frecuente fueron quistes hepáticos que contenían larvas, y lesiones inflamatorias y fibróticas. Conclusión. Estos resultados indican que helmintos de potencial zoonótico circulan en R. norvegicus de ambientes urbanos. Debe investigarse la verdadera distribución de estos parásitos, para determinar el riesgo potencial que corren las poblaciones animales y humanas expuestas a adquirir este tipo de infecciones.

Introduction. Rattus norvegicus, the Norway rat, plays a pivotal role in the maintenance and spread of several zoonotic bacterial, viral and parasitic pathogens of public health interest. The presence of helminthic infections near susceptible human populations can, under appropriate environmental conditions, become a risk factor for their transmission. Objective. Frequencies of infection were reported for Capillaria hepatica and larval forms of Taenia taeniaeformis in wild rats (R. norvegicus) captured in an urban area. Materials and methods. Two hundred and fifty-four adult specimens of R. norvegicus were collected in an urban zone of Medellín, Colombia. The livers of 54 specimens that showed macroscopic hepatic lesions during necropsy were examined by conventional histopathology. Results. The frequency of infestation with C. hepatica was 20.1% (51/254). Six livers (2.4%) were also positive for larvae of T. taeniaeformis. Livers infested with C. hepatica exhibited adult or juvenile parasites and oval eggs with bipolar opercula, and were associated with mild to moderate multifocal granulomatous hepatitis with leucocyte infiltrate. Granulomatous lesions and calcified residual fibroses were found with eggs but without adult parasites. Those animals with cysticerci of T. taeniaeformis showed a high frequency of hepatic cysts containing larvae as well as inflammed and fibrotic lesions. Conclusion. Zoonotic helminths circulate at high frequency in R. norvegicus that occur in urban environments. Further research about the distribution of these parasites will determine the level of health threat they present for susceptible human and domestic animal populations.

[Two cases of disseminated toxoplasmosis]. / Zwei Fallberichte einer disseminierten Toxoplasmose.

Two cases of disseminated fatal toxoplasmosis of a pet mouse and a red panda are described. The pet mouse had a private owner; the red panda lived in a zoological garden in Saxony. At necropsy, both animals suffered from a systemic toxoplasmosis. A severe necrotizing hepatitis was the main histological feature in both animals. Parasitic cysts could be abundantly found in the liver, moderately in the brain and in a low number in other organs. With the PAS-reaction, cysts showed a weak staining in the mouse and a strong staining in the red panda. Diagnosis was confirmed by PCR and immunohistochemistry.

[Rattus norvegicus as an indicator of circulation of Capillaria hepatica and Taenia taeniaeformis on a groceries trade center of Medellín, Colombia]. / Rattus norvegicus como indicador de la circulación de Capillaria hepatica y Taenia taeniaeformis en la Plaza Minorista de Medellín, Colombia.

INTRODUCTION: Rattus norvegicus, the Norway rat, plays a pivotal role in the maintenance and spread of several zoonotic bacterial, viral and parasitic pathogens of public health interest. The presence of helminthic infections near susceptible human populations can, under appropriate environmental conditions, become a risk factor for their transmission. OBJECTIVE: Frequencies of infection were reported for Capillaria hepatica and larval forms of Taenia taeniaeformis in wild rats (R. norvegicus) captured in an urban area. MATERIALS AND METHODS: Two hundred and fifty-four adult specimens of R. norvegicus were collected in an urban zone of Medellín, Colombia. The livers of 54 specimens that showed macroscopic hepatic lesions during necropsy were examined by conventional histopathology. RESULTS: The frequency of infestation with C. hepatica was 20.1% (51/254). Six livers (2.4%) were also positive for larvae of T. taeniaeformis. Livers infested with C. hepatica exhibited adult or juvenile parasites and oval eggs with bipolar opercula, and were associated with mild to moderate multifocal granulomatous hepatitis with leucocyte infiltrate. Granulomatous lesions and calcified residual fibroses were found with eggs but without adult parasites. Those animals with cysticerci of T. taeniaeformis showed a high frequency of hepatic cysts containing larvae as well as inflammed and fibrotic lesions. CONCLUSION: Zoonotic helminths circulate at high frequency in R. norvegicus that occur in urban environments. Further research about the distribution of these parasites will determine the level of health threat they present for susceptible human and domestic animal populations.

Enhanced expression of transforming growth factor-beta 1 in inflammatory cells, alpha-smooth muscle actin in stellate cells, and collagen accumulation in experimental granulomatous hepatitis caused by Toxocara canis in mice.

Although toxocaral granulomatous hepatitis (TGH) characterized with a dominant-Th2 type immune response is a self-limiting disease, little is known concerning the role of fibrosis-related cytokine transforming growth factor-beta 1 (TGF-beta 1) in pathogenesis of TGH. A detailed histological and quantitatively immunohistochemical analysis of TGF-beta 1, alpha-smooth muscle actins (alpha-SMA), and collagen was performed on the liver tissues from mice infected with Toxocara canis as assessed between day 1 and 42 weeks post-infection (DPI or WPI). TGF-beta1 was detected mainly in infiltrating leukocytes in lesions with strong expressions from 4 to 16 WPI. Larvae per se also exhibited strong TGF-beta 1-like molecule expressions in the trial. Alpha-SMA was detected predominantly in hepatic stellate cells (HSC) which surrounded the lesions with moderate expressions largely throughout the period of the entire experiment. Collagen was observed to accumulate in inflammatory lesions and biliary basement with moderate to strong expressions from 1 WPI onwards in the trial. Since many evidences have indicated that leukocytes have the potential to influence HSC by producing TGF-beta 1 which can affect HSC to increase collagen synthesis in various liver diseases, we may propose that persistently elevated TGF-beta 1 expression in infiltrating leukocytes and active HSC with marked alpha-SMA expressions may contribute to healing of injured sites through up-stimulation of collagen deposition; in contrast, abnormally persistent collagen accumulation may cause irreversible fibrotic injury in the TGH.

Feline cholangitis/ chonlangiohepatitis complex secondary to Platynosomum fastosum infection in a cat.

Platynosomiasis has been associated with cholangitis/cholangiohepatitis complex in domestic cats. Despite being typically asymptomatic, some individuals may develop severe disease resulting in hepatic cirrhosis. Definitive diagnosis by means of parasite eggs direct visualization is not always possible and complementary tests should be associated. This paper reports a case of a three-year-old female cat with anorexia and jaundice. Ultrasound and biochemistry laboratory findings as well as liver biopsy were not conclusive. Definitive diagnosis of intense Platynosomum fastosum infestation was only possible through direct examination of biliary fluid at necropsy, stressing the importance of including platynosomiasis, commonly an underdiagnosed disease, among differential diagnoses of feline liver diseases.

[Acute hepatitis cysticercosa and pneumonitis cysticercosa in a lamb: case report]. / Bir kuzuda akut hepatitis cysticercosa ve pneumonitis cysticercosa: olgu sunumu.

The diseases known as hepatitis cysticercosa and pneumonitis cysticercosa are caused by migrating Cysticercus tenuicollis, the intermediate stage of Taenia hydatigena, in the liver and lung tissue of intermediate hosts like sheep, goats, cattle, pigs and squirrels. In this study, parasitological and pathological examinations were performed on the liver and lung tissue samples of a one month old lamb that had died in a herd of Karadayi village, Bünyan, Kayseri. After the examination, hepatitis cysticercosa and parasitic verminous pneumonia (Pneumonitis cysticercosa) were diagnosed and the results were discussed.

Enhanced inducible nitric oxide synthase expression and nitrotyrosine accumulation in experimental granulomatous hepatitis caused by Toxocara canis in mice.

The involvement of inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT) in pathogenesis of toxocaral granulomatous hepatitis (TGH) in a murine host was quantitatively determined by biochemical, parasitological, pathological, and immunohistochemical assessments in a 42-week investigation. Mice were sacrificed for serum collection and histological processing as well as acid-pepsin digestion of the liver in a larval recovery study. Significantly increased levels of total serum NO were found in the trial, indirectly suggesting iNOS activation in the liver. iNOS reactivity was predominantly observed in infiltrating leucocytes in lesions and normal and apocrine-like cholangiocytes; in contrast, hepatocytes and multinucleated giant cells showed negative cytoplasmic staining in TGH. Strong iNOS-like reactivity was also detected on the body wall of larvae. The locations of NT reactivity were nearly identical to those of iNOS expression; infiltrating leucocytes or cholangiocytes stained for iNOS were also stained for NT in TGH. Enhanced iNOS expression, but not invading larvae (r = 0.256, P = 0.211), seemed to play a certain role in pathological damage in TGH due to a significant correlation between iNOS expression and serum alanine aminotransferase (ALT) levels (r =0.593, P = 0.021) in the trial. Our present results indicate a potential therapeutic strategy for treatment of GH caused by other nematodes through manipulation of iNOS expression.

Contribution of CD1d-unrestricted hepatic DX5+ NKT cells to liver injury in Plasmodium berghei-parasitized erythrocyte-injected mice.

Inoculation with erythrocytes infected with Plasmodium berghei, a protozoan causing mouse lethal malaria, induces liver injury in mice, although the parasite cannot invade host hepatocytes at this infectious stage. As previously reported, hepatic infiltrates participate in this liver injury by exerting their perforin-dependent killing action. Here, we have investigated the cellular mechanisms underlying P. berghei-induced incidental liver injury. Hepatic lymphocytes from P. berghei-infected mice killed normal hepatocytes, but not ConA-induced lymphoblasts. Furthermore, the hepatic lymphocytes from infected C57BL/6 mice displayed cytotoxicity against hepatocytes from C57BL/6 and BALB/c mice, indicating MHC-unrestricted hepatocytotoxicity by these hepatic lymphocytes. NK cells were not involved in this hepatocytotoxicity. However, DX5+ cells sorted from the liver of infected CD1d-deficient mice killed normal hepatocytes, indicating that CD1d-independent DX5+ T cells are the effector cells. The hepatocytotoxicity of these hepatic DX5+ T cells did not require TCR engagement. These findings indicate that hepatic CD1d-independent DX5+ T cells play a critical role in P. berghei-induced liver injury. Our studies may have general implications for tissue injuries that are caused by 'bystander killing' or other poorly defined cell-mediated killing mechanisms.

Echinococcus multilocularis in two lowland gorillas (Gorilla g. gorilla).

An unusual presentation of alveolar echinococcosis was observed in two lowland gorillas (Gorilla g. gorilla). Clinical signs included progressive abdominal enlargement, apathy and anorexia. Macroscopical changes consisted of severe peritonitis and foci of hepatic necrosis with large cavities replacing most of the normal tissue. Additionally, a few structures resembling hydatid cysts were present. Histologically, some necrotic areas contained fragments of a laminated wall characteristic of echinococcal metacestodes. Only a few areas showed the multiloculated architecture typical of Echinococcus multilocularis. Serum antibodies against E. multilocularis antigen were detected in both animals, and granulomatous and necrotizing hepatitis with severe peritonitis due to E. multilocularis was diagnosed. The pathological changes in alveolar echinococcosis in gorillas appear to resemble more closely those found in human beings than those in other non-human primates.

Plasmodium berghei infection in mice induces liver injury by an IL-12- and toll-like receptor/myeloid differentiation factor 88-dependent mechanism.

Malaria, caused by infection with Plasmodium spp., is a life cycle-specific disease that includes liver injury at the erythrocyte stage of the parasite. In this study, we have investigated the mechanisms underlying Plasmodium berghei-induced liver injury, which is characterized by the presence of apoptotic and necrotic hepatocytes and dense infiltration of lymphocytes. Although both IL-12 and IL-18 serum levels were elevated after infection, IL-12-deficient, but not IL-18-deficient, mice were resistant to liver injury induced by P. berghei. Neither elevation of serum IL-12 levels nor liver injury was observed in mice deficient in myeloid differentiation factor 88 (MyD88), an adaptor molecule shared by Toll-like receptors (TLRs). These results demonstrated a requirement of the TLR-MyD88 pathway for induction of IL-12 production during P. berghei infection. Hepatic lymphocytes from P. berghei-infected wild-type mice lysed hepatocytes from both uninfected and infected mice. The hepatocytotoxic action of these cells was blocked by a perforin inhibitor but not by a neutralizing anti-Fas ligand Ab and was up-regulated by IL-12. Surprisingly, these cells killed hepatocytes in an MHC-unrestricted manner. However, CD1d-deficient mice that lack CD1d-restricted NK T cells, were susceptible to liver injury induced by P. berghei. Collectively, our results indicate that the liver injury induced by P. berghei infection of mice induces activation of the TLR-MyD88 signaling pathway which results in IL-12 production and activation of the perforin-dependent cytotoxic activities of MHC-unrestricted hepatic lymphocytes.

Granulomatous hepatitis associated with Hepatozoon sp. meronts in a southern water snake (Nerodia fasciata pictiventris).

A wild-caught adult female southern water snake (Nerodia fasciata pictiventris) did poorly in captivity. A peripheral blood-film examination demonstrated numerous hemogregarines characterized as fusiform nondividing intraerythrocytic gametocytes. Xenodiagnostic typing in laboratory-reared mosquitoes demonstrated the parasite to be of the genus Hepatozoon. Gross and histopathologic examination of the liver demonstrated numerous granulomas centered on groups of one to six Hepatozoon sp. meronts, an unusual finding in naturally infected wild-caught snakes.

Eosinophilic colitis and hepatitis in a horse with colonic intramucosal ciliated protozoa.

Tissues from a 9-year-old American Standardbred gelding with a history of anorexia, mild colic, and unexpected death were submitted to the Laboratories of Veterinary Diagnostic Medicine at the University of Illinois for histopathologic examination. Microscopic diagnoses were severe subacute, diffuse eosinophilic colitis with intralesional protozoa and a subacute to chronic eosinophilic portal hepatitis with granuloma formation. Two tissue-invading, ciliated protozoa were identified in large numbers within the colonic mucosa. The ciliates were Polymorphella ampulla and Cycloposthium sp., Phylum:Ciliophora. The eosinophilic colitis and portal hepatitis in association with the overpopulation and invasion by the ciliated protozoa suggests a causal etiology.

A survey and review of hepatobiliary lesions in Australian macropods.

This report presents information on the range of diseases and lesions that occurred in sections of livers of macropods held in the Non-Domestic Animal Registry at Taronga Zoo. Of 142 affected livers, 52 were due to parasites, 24 to bacteria, nine to Macropod Herpesvirus, four to fungal agents and ten to tumours. In addition, 17 livers had acute degenerative or necrotic lesions, 22 were affected by fibrosis or other chronic lesions and four had miscellaneous degenerative lesions. Common parasitic diseases included toxoplasmosis, coccidial cholangitis, cestode cholangitis (Progamotaenia sp.), fascioliasis and capillarial hepatitis. Bacterial diseases were varied but lesions due to anaerobic bacteria or Mycobacteria spp. were most common. Many of the acute degenerative lesions were associated with cardio-respiratory disease. Chronic lesions were often of unknown origin, although it was speculated that parasitic and bacterial organisms contributed to the aetiology. There were four primary and six metastatic tumours.

First report of infectious necrotic hepatitis (black disease) among Nubian goats in Sudan.

In a flock of 425 female and male Nubian goats in the Khartoum Province, an outbreak of a disease causing sudden death of 18 apparently healthy goats occurred (11 females and 7 males, 3-6 years old). Adult Fasciola gigantica were found in the livers of all goats and in seven of them Cysticercus tenuicollis cysts. These organs showed necrotic and severe histopathological changes. Clostridium novyi type B was isolated from necrotic areas of all livers and found to be highly pathogenic and toxigenic to laboratory animals. The disease was diagnosed as infectious necrotic hepatitis (black disease). Faecal examination revealed the presence of F. gigantica eggs. Lymnaea natalensis snails were found to be prevalent in the water canals. As the Khartoum Province is regarded as an endemic area for black disease, routine vaccination is highly recommended for its control in goats and sheep.