Duplex nested-PCR for detection of small ruminant lentiviruses

Abstract Small ruminant lentiviruses (SRLV) have high genetic variability which results in different viral strains around the world. This create a challenge to design sensible primers for molecular diagnosis in different regions. This work proposes a protocol of duplex nested-PCR for the precise diagnosis of SRLV. The technique was designed and tested with the control strains CAEV Co and MVV 1514. Then, field strains were submitted to the same protocol of duplex nested-PCR. Blood samples of sheep and goats were tested with AGID and nested PCR with specific primers for pol, gag and LTR. The AGID results showed low detection capacity of positive animals, while the nested PCR demonstrated a greater capacity of virus detection. Results demonstrated that LTR-PCR was more efficient in detecting positive sheep samples, whereas gag-PCR allowed a good detection of samples of positive goats and positive sheep. In addition, pol-PCR was more efficient with goat samples than for sheep. Duplex nested PCR performed with standard virus samples and field strains demonstrated that the technique is more efficient for the detection of multiple pro-viral DNA sequences. This study demonstrated a successful duplex nested PCR assay allowing a more accurate diagnosis of SRLV.

Virus orf en humanos, confirmación molecular de un caso clínico en Chile / Orf virus in human, confirmation in case report from Chile

Resumen La infección por el virus orf, también conocida como ectima contagioso, es reconocida una zoonosis ocupacional. Se diagnostica por lesiones cutáneas que evolu cionan rápidamente desde máculas a pápulas, vesículas y pústulas. Se presenta el caso clínico de una estudiante de medicina veterinaria que había tenido contacto con caprinos, clínicamente sanos y sin lesiones aparentes, hacía 19 días. Presentó dos lesiones vesiculares que coalescieron hasta formar una lesión de mayor tamaño rodeada por un halo eritematoso. Las lesiones fueron compatibles con la presentación clásica de las producidas por el virus orf en humanos. Se confirmó la presencia del virus orf mediante una RPC anidada del tejido de biopsia. Es uno de los primeros casos confirmados mediante técnicas moleculares en seres humanos en Chile.

Infection with the orf virus, also known as contagious ecthyma, is recognized as an occupational zoonosis worldwide. It is diagnosed by cutaneous lesions that progress rapidly from macules to papules, vesicles and pustules. The clinical case of a student of veterinary medicine who had had contact with goats, clinically healthy and without apparent lesions, which occured 19 days ago, is reported. She presented two vesicular lesions that coalesced to form a larger lesion surrounded by an erythematous halo. The lesions were compatible with the classical presentation of those produced by the orf virus in humans. The presence of the orf virus was confirmed by a nested PCR from biopsy tissue. It is one of the first cases confirmed by molecular techniques in humans in Chile.

Interspecific transmission of small ruminant lentiviruses from goats to sheep

This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.

The Gut and Lung Morphometry in Experimental and Natural Lineage 1 Variant of Peste des Petits Ruminants Virus Infection in Nigerian Goats / Morfometría Intestinal y Pulmonar en la Infección Natural y Experimental del Virus Peste des petits ruminants del linaje 1 en Cabras de Nigeria

The lung and gut morphometry in both natural and experimental Peste de petit ruminant (PPR) virus which are scanty in literature hence the need for this study. The goats that were submitted for necropsy in the Department of Veterinary Pathology University of Ibadan between 2009 and 2010 and the gross pathological diagnosis were PPR were enrolled in this study. The degree of pneumonia as a percentage of the total lung volume was estimated using standard methods. The gut morphometry of goats experimentally infected with PPR virus was also used. Student "T" test was used for the test of significance in evaluating the effect of age, sex and the lung consolidation pattern in natural PPR and analysis of the gut morphometry. Complicated PPR had significant higher pulmonary consolidation when compared with the uncomplicated PPR (p< 0.05). The pulmonary consolidation was significantly higher on the right lung with a mean percentage value of 6.54 than the left lung (p< 0.05). The caudal lobe was more consolidated than the cranial and middle lobes in natural PPR. The pulmonary consolidation was more in goats less than a year, while the buck had a significantly higher pulmonary consolidation than the does (p< 0.05). There was no significant difference in the mean length of the villi and width of the villi of PPR virus infected goats when compared to the control, however a significant difference was observed in the cryptal depth (p< 0.05). There was a significant difference in the mean villi length and cryptal depth of goats with complicated PPR (Mannheimia hemolytica) infected goats (p< 0.05) relative to the control. From this study, it showed that most natural PPR were complicated with bacteria and this complication may have contributed to the fatality associated with PPR especially those caused by lineage 1 viruses. This study also showed that secondary bacterial involvement in course of PPR affect the gut morphometry and that could account for the severity of intestinal lesion commonly observed with field PPR in Nigerian goats.

La morfometría del pulmón y el intestino en la infección del virus Peste des petits ruminants (PRR) de forma natural así como experimental es escaza en la literatura, de ahí la necesidad de este estudio. Fueron incluidas en este estudio las cabras que fueron sometidas a autopsia en el Departamento de Patología Veterinaria de la Universidad de Ibadan entre 2009 y 2010, con diagnóstico patológico macroscópico de PPR. El grado de neumonía como porcentaje del volumen pulmonar total fue estimado mediante los métodos estándar. También fue determinada la morfometría del intestino de las cabras infectadas experimentalmente con el virus PPR. Se utilizó la prueba "T" de Student para determinar la significancia en la evaluación de los efectos de edad, sexo, patrón de consolidación pulmonar en PPR natural y análisis de la morfometría intestinal. La PPR complicada tuvo una consolidación pulmonar altamente significativa en comparación con la PPR no complicada (p <0,05). La consolidación pulmonar fue significativamente mayor en el pulmón derecho, con un valor porcentaje promedio de 6,54 en comparación al pulmón izquierdo (p <0,05). El lóbulo caudal fue más consolidado que los lóbulos craneal y medio en presencia del PPR natural. La consolidación pulmonar fue más frecuente en caprinos menores de un año, mientras que los machos cabríos tuvieron una consolidación pulmonar significativamente más alta (p <0,05). No hubo diferencias significativas en la longitud y ancho promedio de las vellosidades en cabras infectadas con PPR en comparación con el control, pero se observó una diferencia significativa en la profundidad de las criptas (p <0,05). Hubo diferencia significativa en la longitud de las vellosidades y la profundidad media de las criptas en las cabras infectadas con PPR complicada (Mannheimia haemolytica) (p <0,05) en relación al control. A partir de este estudio, se demostró que las infecciones con PPR natural se complicaron con bacterias, y estas complicaciones pueden haber contribuido a la mortalidad asociada el PPR, especialmente las causadas por el virus del linaje 1. Este estudio también mostró que la participación bacteriana secundaria en el curso de la PPR afecta la morfometría intestinal y que podría dar cuenta de la gravedad de la lesión intestinal observada comúnmente en la infección de PPR en cabras de Nigeria.

The pattern of distribution of pneumonic consolidation in experimental peste des petits ruminants virus (PPRV) and/or Mannheimia Hemolytica Infection in West African dwarf goats / Patrón de distribución de la consolidación pneumónica en infección experimental con virus peste des petits ruminants (PPRV) y/o mannheimia hemolítica en cabras enanas del Oeste Africano

The study into the pattern of distribution of the lung consolidation associated with common viral and bacterial pneumonia and their co-infection in subsaharan goats is scanty in literatures. Fifty apparently healthy West Africa Dwarf goats (WAD) six months of age were used for the experiment. The animals were divided into groups A, B, and C with 15 goats each while 5 goats served as control. Group A goats infected with 1ml of pure culture (1 X 109 CFU) of Mannheimia haemolytica MH A2, while group B with 1ml of pure cultured 106.5 TCID50 PPR virus grown in Baby hamster kidney cell lines and group C with 1 ml of PPRV and a week later 1ml of MH A2. The degree of consolidation or pneumonia as a percentage of the total lung volume was determined by visual observation, palpation and measurement of the lesion which is estimated as a percentage of each lobe. Student t-test were used to test for significant differences. The right lungs have a higher lung consolidation percentage than the left in all the treatment groups. The accessory lobe was affected in the PPRV group. The MH group has the highest lung consolidation percentage (10.1 percent). The PPRV 1-28dpi has the lowest consolidation percentage (1.06 percent). There is significant difference in the consolidation percentage and mortality between MH, PPR+MH, PPRV 28-45 dpi and PPRV 1-28dpi (P<0.05). This observation further show that the right lung and the anterior lobes were more affected in experimental viral and bacterial respiratory pathogen and their co-infection as the trachea birfucation is first to the right and the distance between the right and the left birfucation was 1.5 +/- 0.35cm. It is the first study that describes and compare the pattern of distribution and morphometry of pneumonia in experimental PPRV, MH and PPRV+MH infections in goats.

El estudio sobre el patrón de distribución de la consolidación pulmonar asociada con neumonía virales y bacterianas comunes y sus co-infección en cabras Subsaharianas, es escasa en la literatura. Cincuenta cabras enanas de África occidental (WAD) aparentemente sanas de seis meses de edad fueron utilizados para el experimento. Los animales se dividieron en grupos A, B y C con 15 cabras cada uno mientras que el 5 cabras sirvió como control. Grupo A cabras infectadas con 1 ml de cultivo puro (1 X 109 UFC) de Mannheimia haemolytica MH A2, mientras que el grupo B con 1 ml de cultivo puro 10 6,5 DICT50 PPR cultivado en líneas celulares de riñón de crías de hámsters y el grupo C con 1 ml de PPRV y un semana después de 1 ml de MH A2. El grado de consolidación o neumonía como porcentaje del volumen pulmonar total se determinó por observación visual, palpación y la medición de la lesión que se estima como un porcentaje de cada lóbulo. El test t de Student se utilizaron para probar las diferencias significativas. El pulmón derecho tiene un porcentaje de consolidación pulmonar superior a izquierdo en todos los grupos de tratamiento. El lóbulo accesorio se vio afectado en el grupo de PPRV. El grupo MH tiene el porcentaje más alto de consolidación pulmonar (10,1 por ciento). El PPRV 1-28dpi tiene el menor porcentaje de consolidación (1,06 por ciento). No hay diferencia significativa en el porcentaje de consolidación y la mortalidad entre MH, MH + PPR, PPRV 28-45 dpi y PPRV 1-28dpi (P <0,05). Esta observación muestra además que el pulmón derecho y los lóbulos anteriores se vieron más afectados en infecciones respiratorias patógenas experimentales con agentes virales y bacterianos y su co-infección como la bifurcación traqueal es primero a la derecha y la distancia entre la derecha y la bifurcación izquierda fue de 1,5 +/- 0,35 cm. Es el primer estudio que describe y compara el patrón de distribución y la morfometría de las neumonías en PPRV experimentales, MH y MH + PPRV...

Histological changes induced by viral hepatitis, colitis and nephritis-cystitis in goats at Gassim Region, central Saudi Arabia

Histological lesions of viral hepatitis, viral colitis and viral nephritis-cystitis, characterized by presence of intracytoplasmic inclusion bodies in involved tissues, were seen in goats that died of acute unrecognized diseases. The viral inclusion bodies stained positively by special staining for viral nucleic acid. Viral hepatitis was diagnosed in an adult goat which experienced a fulminating acute disease. Post-mortem examination showed that the liver was swollen and dark in color. Histopathological examination revealed severe congestion, fatty change and necrosis of the liver cells as well as presence of eosinophilic intracytoplasmic inclusion bodies in the hepatocytes. Viral colitis was diagnosed in 3 kids that died of severe haemorrhagic diarrhoea. The disease occurred in a small farm containing about 75 kids, 22 of which died of clinical disease. None of the adult animals showed clinical signs. The lesions were confined to the colon which was haemorrhagic and full of clotted blood. Histopathological examination showed haemorrhagic ulcerative colitis and presence of acidophilic intracytoplasmic inclusion bodies in the mucosal epithelium. Bacteriological examination showed presence of enterotoxogenic Echerichia coli. Viral nephritis-cystitis was diagnosed in a male kid of about 4 months old. The animal developed acute disease with bloody urine and died within one day of appearance of clinical signs. The lesions were confined to the urinary system and that both renal pelvis and urinary bladder were full of clotted blood. Extensive haemorrhage was present in the renal cortex and medulla. Histopathology revealed necrosis of both renal tubules and glomeruli, haemorrhagic cystitis and presence of intracytoplasmic inclusion bodies in the transitional epithelium of the urinary bladder and glomerular lining. Bacteriological examination was negative for leptospirosis

Prevalência da infecçäo pelo vírus da artrite encefalite caprina no Estado do Ceará, Brasil / Prevalence of caprine arthritis encephalitis virus infection in the state of Ceará, Brazil

A elevaçäo do risco de contaminaçäo pelo vírus da artriteencefalite caprina (CAEV) em rebanhos caprinos nativos e sem raça definida (SRD) tem sido verificada paralelamente à introduçäo de caprinos de raças exóticas no Brasil, o que motivou a realizaçäo deste levantamento epidemiológico no Estado do Ceará. Para o diagnóstico da infecçäo pelo CAEV, utilizou-se a microtécnica de imunodifusäo em gel de ágar. Foram pesquisadas 4019 amostras de soro caprino em 30 municípios do Ceará. A prevalência da infecçäo pelo CAEV verificada foi de 1 por cento (40/4019 animais), sendo a maior prevalência (11,1 por cento) na regiäo metropolitana de Fortaleza. Analisando a distribuiçäo de animais soropositivos nos municípios pesquisados, constatou-se que um terço dos municípios (10/30) apresentaram pelo menos um animal soropositivo. Verificou-se maior prevalência (p<5 por cento) nos caprinos mais velhos quando comparados com os jovens. A análise por sexo e composiçäo do rebanho mostrou que os machos estavam estatisticamente mais afetados (p<5 por cento). Consideramdo-se o grau de sangue, 5,02 por cento de raças puras e 0,12 por cento de animais mestiços apresentavam anticorpos contra CAEV. Dentre os soropositivos, a raça Parda Alpina foi estatisticamente mais afetada (p<5 por cento). Com base nos resultados, pode-se verificar que o lentivírus de pequenos ruminantes já se encontra disseminado nas várias regiöes do Ceará e que, provavelmente, os reprodutores sejam a principal fonte de transmissäo viral entre os rebanhos nativos/SRD.

Evidência sorológica de infecçäo pelo vírus da artrite-encefalite caprina em caprinos leiteiros do Estado de Pernambuco / Sorological evidence of caprine arthritis-encephalitis in dairy goats in Brazil

Foram pesquisados anticorpos precipitantes contra o vírus da artrite-encefalite caprina (CAE), empregando-se a imunodifusäo em ágar-gel, utilizando-se o antígeno do vírus maedi/visna dos ovinos, em 214 amostras de soros de caprinos puros (Saaneu, Toggenburg e Parda Alpina) e seus mestiços, provenientes de cinco exploraçöes leiteiras de quatro municípios do Estado. Evidenciaram-se resultados positivos em 38 amostras (17,7 por cento), com frequências por criaçäo variando de zero a 29,5 por cento