María Jesús Sánchez Muñoz

Role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in Spain

Faeces samples from diarrhoeic and non-diarrhoeic lambs and goat kids aged 1-45 days were examined for enteric pathogens. Cryptosporidium parvum was detected in both diarrhoeic lambs (45%) and goat kids (42%) but not in non-diarrhoeic animals. F5+ (K99+) and/or F41+ Escherichia coli strains were isolated from 26% and 22% of the diarrhoeic lambs and goat kids, respectively, although these strains, which did not produce enterotoxins ST I or LT I, were found with similar frequencies in non-diarrhoeic animals. A F5-F41-ST I+ E. coli strain was isolated from a diarrhoeic lamb (0.6%). Verotoxigenic E. coli was isolated from both diarrhoeic and non-diarrhoeic lambs (4.1% and 8.2%, respectively) and there was no association between infection and diarrhoea. The prevalence of group A rotavirus infection in diarrhoeic lambs was very low (2.1%). Groups A and B rotaviruses were detected in three (8.1%) and five (13.5%) diarrhoeic goat kids from two single outbreaks. Group C rotaviruses were detected in four non-diarrhoeic goat kids. An association of diarrhoea and infection was demonstrated only for group B rotavirus. Clostridium perfringens was isolated from 10.8% of the diarrhoeic goat kids but not from non-diarrhoeic goat kids or lambs. Salmonella arizonae was isolated from a diarrhoeic goat kid (2.7%) and the clinical characteristics of the outbreaks where these two latter enteropathogens were found different from the rest. Picobirnaviruses were detected in a diarrhoeic lamb. No coronaviruses were detected using a bovine coronavirus ELISA. No evidence was found of synergistic effect between the agents studied. Enteric pathogens were not found in four (8.7%) and three (20%) outbreaks of diarrhoea in lambs and goat kids, respectively.

Variation in the Immuno-pathological Responses of Lambs after Experimental Infection with Different Strains of Mycobacterium avium subsp. paratuberculosis

Ruminant infection by Mycobacterium avium subsp. paratuberculosis (MAP) causes a granulomatous inflammatory response in the intestine and associated lymph nodes. Differences either in the affected organs or in the inflammatory infiltrate were observed between species and individuals. Such differences are usually attributed to variations in host immune responses or to inconsistent effects among different MAP strains. To evaluate if different MAP strains induce different immuno‐pathological responses in lambs, 28 one‐month‐old individuals were divided into six groups and inoculated with different MAP strains. Groups 1 and 2 were inoculated with two bovine strains isolated in Argentina that showed different genetic patterns after BstEII‐IS900‐RFLP (hereafter strains E and A respectively). Group 3 was inoculated with a bovine strain isolated in Spain obtained after a previous step of culture (patterns C1). Group 4 was inoculated with a homogenate of intestinal mucosa of a clinical case affected by the same bovine strain as that of group 3. Group 5 was inoculated with an ovine strain that was directly purified from the intestinal mucosa of a clinical case, and group 6 was kept as control (i.e. no inoculation). Peripheral immune responses were assessed until 150u2003days post‐infection (dpi), when lambs were humanely killed. Pathological studies were performed in tissues from the intestine and lymph nodes. Lesion types and inflammatory infiltrates were examined as indicators of pathogenicity. All the lambs infected with bovine MAP strains showed a common lesion pattern regardless of the strain type. Such pattern was characterized by focal lesions mainly in the mesenteric lymph nodes, the presence of fibrous tissue, and, occasionally, necrosis in the granulomas as well as the presence of numerous giant cells. Differences in lesion severity were observed among groups: lambs from groups 1 and 2 had the highest number of granulomas and the largest lymph node area affected. Lesions in animals from group 5 (infected with an ovine strain) were more severe and occurred mostly in the intestinal lymphoid tissue; necrosis, fibrosis or giant cells were never detected in this group. These results indicate that the MAP strain type induces different pathological responses in lambs.

Nephrotoxicosis in Iberian Piglets Subsequent to Exposure to Melamine and Derivatives in Spain between 2003 and 2006

Between November 2003 and September 2006, 300 to 400 45–60-day-old Iberian piglets developed anorexia, polydipsia, and lethargy. Piglets were from 5 different farms in the western part of Spain. Morbidity was between 40% and 60%, and mortality ranged from 20% to 40% of the total population of postweaning piglets. In the 9 piglets in which postmortem examinations were conducted, kidneys were enlarged with yellow foci in the cortex and medulla. Microscopically, these foci were accumulations of crystals within the lumina of dilated distal tubules and collecting ducts, causing flattening of the renal tubular epithelial cells. The crystals displayed a multicolored birefringence under cross-polarized light. The multinucleated giant cells surrounding the crystals, interstitial fibrosis, and nonsuppurative infiltrates indicated a chronic inflammatory response. Toxicologic analysis of fixed kidney tissues from 4 piglets demonstrated the presence of melamine, ammeline, ammelide, and cyanuric acid. Ammelide concentrations were highest, ranging from 39,000 to 92,000 mg/kg, followed by ammeline (20,000–34,000 mg/kg), melamine (9,200–29,000 mg/kg), and cyanuric acid (2,200–9,100 mg/kg). The clinical, histologic, and toxicologic findings in affected piglets were similar to those reported in dogs and cats that died of melamine and melamine analogue-associated renal failure in 2004 and 2007. To the authors knowledge, this is the first documented report of poisoning due to melamine and its analogues in pigs and demonstrates that contamination of pig feed occurred as early as 2003.

An outbreak of diarrhoea associated with atypical rotaviruses in goat kids.

The presence of rotaviruses was investigated in the faeces of 31 goat kids in a dairy herd that experienced an outbreak of severe diarrhoea which caused dehydration, anorexia and prostration in seven (22.6 per cent) of them. All the affected animals were two to three days old. A group A-specific ELISA failed to detect rotaviruses in any of the samples but the characteristics electropherotype of group B rotaviruses was observed by electrophoresis in polyacrylamide gels in six of the animals. A highly significant statistical association between the shedding of rotavirus and the occurrence of diarrhoea was demonstrated. All the rotaviruses were detected in animals three to four days old. Cryptosporidium parvum, Clostridium perfringens and enterotoxigenic Escherichia coli were not detected in the seven diarrhoeic animals.

Rotavirus excretion by kids in a naturally infected goat herd

n Abstractn n A cross-sectional study was carried out in a dairy goat herd, investigating the presence of rotavirus by means of ELISA, polyacrylamide gel electrophoresis (PAGE) and two latex agglutination tests in feces of 63 goat kids younger than 1 month, with and without diarrhea, and in feces of 19 adult goats during the first few days after parturition. All animals belonged to a herd located in the mountains of the León province (NW Spain). Rotaviruses were found in 18 out of 63 goat kid fecal samples but no significant association between shedding of rotavirus and presence of diarrhea could be established. Rotaviruses were found in kids aged 6 to 21 days, and more frequently between 6 and 10 days. No shedding of virus was detected in any of the adults. Considering ELISA as the reference test, PAGE and both latex agglutination tests were less sensitive. One of the latex tests was also highly non-specific. All PAGE-positive samples showed the typical electropherotype of group A rotavirus. Feces were also screened for other pathogens including Escherichia coli, Clostridium perfringens and Cryptosporidium parvum. C. parvum oocysts were detected in the feces of six out of 45 goat kids tested, all six suffering from diarrhea. This paper represents the first description of rotavirus infections in goats in Spain. The possible mechanisms of viral diffusion within the herd and its role as pathogen in goats are discussed.n n

Expression of transforming growth factor-beta 1 (TGF-β1) in different types of granulomatous lesions in bovine and ovine paratuberculosis

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is manifested by a broad spectrum of clinical and lesional presentations. We have evaluated the expression of transforming growth factor-beta1 (TGF-beta1), a cytokine known to have immunosuppressor effects, by immunohistochemistry, in different paratuberculosis lesions in the intestine and lymph nodes from 20 sheep and 25 cattle. Peripheral immune responses were assessed by interferon-gamma (IFN-gamma) test and the presence of antibodies. Expression of TGF-beta1, observed in macrophages and giant cells forming the lesions, was closely related to the amount of Map. In focal and multifocal forms, usually positive to IFN-gamma test, bacilli were difficult to detect and TGF-beta1 expression was low or absent. Diffuse multibacillary lesions, negative to IFN-gamma, show large numbers of Map and the highest percentage of immunolabelled cells. Diffuse paucibacillary forms, positive to IFN-gamma, have low numbers of AFB and scant or no cells positive to TGF-beta1. The high expression of TGF-beta1 would be related to the inability of macrophages to limit the multiplication of Map.

Prevalence of neutralizing antibodies to 9 rotavirus strains representing 7 G-serotypes in sheep sera

Neutralizing antibodies to 9 rotavirus strains representing serotypes G1, G3, G5, G6, G8, G9, and G10 were investigated in 212 ovine serum samples from 3 age groups, 1-week-old lambs, 2- to 3-months-old lambs and adult sheep. All sera from 1-week-old lambs had neutralizing antibodies to all 9 rotavirus strains. Both neutralizing antibody titers and prevalences to all 9 strains markedly decreased in the 2- to 3-months-old lamb group and increased again in the adult sheep group. Also, adult sheep sera neutralized a larger number of rotavirus strains than 2- to 3-months-old lamb sera. The highest neutralizing antibody titers and prevalences were found to strains B223 and K923, representing serotype G10, to strain RRV, representing serotype G3, and to strain NCDV, representing serotype G6, indicating that these could be the predominant 3 rotavirus serotypes in Spanish sheep. The rotavirus serotypes infecting sheep observed by us differ from those described for cattle, where G6 is the most prevalent serotype followed by G10, and G3 has been seldom found. Very low prevalences were observed for strains WA and OSU representing serotypes G1 and G5 respectively, suggesting that they probably do not infect sheep and neutralizing antibodies found are derived from heterotypic responses to other serotypes. Intermediate prevalences and titers were found to strains UK (serotype G6), 69M (serotype G8) and WI61 (serotype G9). Neutralizing antibodies distinguished between different strains sharing their VP7 specificity: B223 and K923, a bovine and an ovine serotype G10 strains, and NCDV and UK, two serotype G6 bovine rotavirus strains with different VP4 antigen.

Molecular and Serologic Characterization of a Group a Bovine Rotavirus with a Short Genome Pattern

Rotaviruses are members of the family Reoviridae and are a widespread cause of diarrhea in the young of many mammalian species, including humans. The rotavirus particles are nonenveloped and possess an icosahedral symmetry. The genome consists of 11 discrete segments of linear doublestranded RNA (dsRNA) and is enclosed within a doublelayered capsid. The dsRNA fragments are numbered 1 to 11 on the basis of their order of migration during polyacrylamide gel electrophoresis (PAGE). Rotaviruses are classified into serotypes based on the specificity of the outer capsid proteins VP7 (G types) and VP4 (P types) The more common method of serotyping is on the basis of G types (13), and currently 14 G types have been identified among group A rotaviruses. Among group A bovine rotaviruses (BRVs), at least 4 G types (G1, G6, G8, G10) have been reported based on the results of enzymelinked immunosorbent assay (ELISA), virus neutralization assays, nucleic acid hybridization assays, or sequence analysis. Rotaviruses are also typed using similar procedures on the basis of VP4 specificity, and currently there are at least 4 P types of BRV (P1, P5, P11, and P12). Upon analysis by PAGE, the 11 dsRNA segments of rotaviruses produce characteristic patterns referred to as genome electropherotypes. 35 Such electrophoretic analysis of BRV isolates is useful for obtaining epidemiologic information regarding the origin of the isolates, because each isolate produces a unique pattern. There have been numerous reports of short genome electropherotypes among human rotaviruses. Most of these short electropherotypes are restricted to subgroup I and share serotype G2 specificity. All BRV isolates tested so far, regardless of genome electropherotype, belong to subgroup I, and most possess long electropherotypes.38 Recently, there have been reports of short electropherotypes among BRV isolates, all restricted to serotype G6 1,22,37 Because there have been no reports of non-G6 BRV with a short genome pattern, we wanted to investigate the possibility that BRV short pattern isolates are restricted to serotype G6, similar to the situation observed for human G2 rotaviruses. In addition, we wanted to correlate the hybridization data with serology and cross-protection studies. In this report, we describe the serologic and molecular characterization (G and P specificity) of a bovine rotavirus strain, 2292B, with a short genome pattern. The reference viruses were grown in rhesus monkey kidney (MA104) cells in roller bottles and titrated by a plaque assay as previously described. The sources and serotypes (P and

Seroprevalence of porcine respiratory coronavirus infection in Spanish breeding sows

n Abstractn n The seroprevalence of porcine respiratory coronavirus (PRCV) among breeding sows was estimated in Castilla y León, the largest region in Spain, by a cross-sectional study. Serum samples (1247) were taken from sows from 58 different herds for this purpose throughout 1988 and were tested by a monoclonal antibody-capture ELISA (MACELISA), a test which detects antibodies to both transmissible gastroenteritis virus (TGEV) and PRCV. In order to discriminate positive sera to both coronaviruses, a blocking inhibition ELISA was used for all MACELISA-positive sera. By MACELISA, 31.4% of sera tested were positive and all of them were confirmed specifically as PRCV-positive by blocking inhibition ELISA, thus ruling out the presence of TGEV in the sampled area: 64% of the farms had at least one PRCV-seropositive sow, indicating that the infection was widespread Farms were classified into three categories, according to size and management practices. No significant differences were found in the prevalence rates among the three farm types; there was, however, a highly significant correlation between increasing farm size and increasing within-farm seropositivity.n n

Detection of Serotype G6 Rotaviruses in Bovine Field Samples Using a Nonradioactive PCR-Derived cDNA Probe

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