Diagnostic value of intravenously administered johnin purified protein derivative (PPD) in bovine paratuberculosis

The diagnostic value of intravenously administered johnin purified protein derivative (PPD) was studied in 45 cattle of different age, coming from herds infected by, or free from, Mycobacterium paratuberculosis. In addition to observing the clinical symptoms, the animals' sera were assayed for specific antibodies by the complement fixation (CFT) and immunodiffusion (AGID) tests. The blastogenic transformation of peripheral lymphocytes was determined on the basis of 3HTdR incorporation. Changes in the neutrophilic leucocyte/lymphocyte ratio of the blood were also monitored. Detection of the pathogen in the faeces was attempted by microscopic examination and by culturing. Combined evaluation of responses elicited by intravenously administered johnin PPD can be a valuable aid in recognizing infected animals, particularly those among the heifer progeny of infected cows.     

The epidemiology of paramphistomosis of sheep (<Emphasis Type="Italic">Ovis aries</Emphasis> L.) in the north west temperate Himalayan region of India

An epidemiological study with the objective to assess the prevalence of paramphistomosis in association with season, age, sex and breed was carried out in naturally infected sheep over a period of two years from February 2005 to January 2007. Gastrointestinal tract (GIT) and faecal examination were conducted monthly to monitor the seasonal occurrence of paramphistomosis. 793 sheep were examined in the first year, out of which 7.06% were positive for Paramphistomum infection. In the second year, 740 animals were investigated and 7.7% were infected. The overall prevalence of paramphistomosis was 7.3% with a mean of 56.50 ± 0.50 and 95% confidence interval (CI) (lower bound: 50.1469; upper bound: 62.8531). The prevalence of paramphistomosis through GIT examination (P = 0.593) was 7.6% at 95% CI (lower bound: −19.1186; upper bound: 57.1186) and the prevalence through faecal examination (P = 0.884) was 7.2% at 95% CI (lower bound: 5.7345; upper bound: 69.2655). Generally, season and age were the factors found to have a significant influence on the risk of paramphistomosis in sheep. The highest infection was found in the summer season (P < 0.005); lower age groups (P < 0.005) in males and in migratory (Bhakarwal) breed (P ≥ 0.005). Winter, adult animals, females and local breed reported low infection. The present study will be of great significance to understand the epidemiology of gastrointestinal helminthes of sheep initially in the resource poor communities of Himalayan region and will definitely be helpful to devise appropriate control strategies for paramphistomosis.     

Spontaneous murine thymocyte comitogenic activity consistent with interleukin-1 in cattle naturally infected with Mycobacterium paratuberculosis

The production of comitogenic activity consistent with interleukin-1 (IL-1) activity by blood monocytes from cattle with naturally acquired paratuberculosis was examined by murine thymocyte proliferation. In addition, IL-1-like activity in response to homologous and heterologous antigens was determined. Activity was determined in nine cattle naturally infected with Mycobacterium paratuberculosis and six non-infected cattle. Comitogenic properties were measured in response to M. paratuberculosis antigen (johnin), bacterial lipopolysaccharide (LPS) as a positive control, and culture media as a negative control. Monocytes from infected cattle spontaneously released high levels of IL-1-like activity in the absence of stimuli and significantly (P less than 0.05) increased activity in response to LPS. With johnin, M. bovis PPD and KLH stimulation, comitogenic activity was similar to spontaneous levels. Non-infected cattle had significantly (P less than 0.05) increased comitogenic activity when blood monocytes were stimulated with KLH, M. bovis PPD, johnin, and lipopolysaccharide when compared with non-stimulated cells in that group. Johnin produced the greatest response in non-infected animals. The data suggest that blood monocytes in infected cattle are non-specifically activated with respect to IL-1 production. Alternatively, a defective regulatory mechanism for IL-1 may be operative in infected cattle. In addition, the previous observation that mycobacterial antigens are potent inducers of IL-1 was also verified.     

Studies of infectivity and pathogenicity of an isolate of Trypanosoma evansi in Yankasa sheep.

The course of experimental infection and pathogenicity of an isolate of Trypanosoma evansi were investigated using eight infected and six uninfected control Yankasa sheep. The sheep were each infected intravenously via the jugular vein with approximately 2.0 x 10(6) T. evansi parasites. The effects of the parasite on body temperature, packed cell volume (PCV), haemoglobin, erythrocytes, total protein, were monitored three times a week for approximately 9 weeks. Body weights were determined once every week for the duration of the experiment. The results showed that all the infected sheep were positive for the parasite. The prepatent period varied between 3 and 6 days. T. evansi produced parasitaemic waves at an average of 8.3 days interval. Two distinct forms of the disease were produced namely, acute (4-14 days postinfection), and chronic (43-59 days postinfection). Anaemia was a distinct feature of the disease. While the mean rectal temperatures were significantly elevated (P < 0.05), the mean values of the haematological parameters of the infected sheep dropped significantly (P < 0.05) compared to the preinfection levels. Observed clinical signs included pale mucous membrane, epiphora, loss of appetite, emaciation, dullness and rough hair coat together with fluctuating pyrexia which in most cases coincided with rise in parasitaemia. It is suggested that the isolate of T. evansi is pathogenic for Yankasa sheep.     

Serological survey of antibodies to <Emphasis Type="Italic">Ehrlichia ruminantium</Emphasis> in small ruminants in Tanzania

Sera from 497 sheep and 555 goats collected in a cross sectional study from different geographical locations in north-eastern Tanzania were examined for antibodies to Ehrlichia ruminantium using MAP 1-B ELISA technique. E. ruminantium antibodies were found in 68.6% (341/497) of sheep and 64.7% (359/555) of goats. Overall seroprevalence was 66.5% (700/1052). Infection rates were higher in sheep than goats (P < 0.05), in pastoral than in agro-pastoral production systems (P < 0.05) and in female sheep than males (P < 0.05). (131/143) 91.6% of the farms/flocks tested revealed sero-positive animals. E.ruminantium infections were found in all the geographical villages and districts tested. The infection rates per administrative district varied from 36.4% (Muheza) to 90% (Mkinga) in goats and from 11.9% (Muheza) to 94.6% (Mkinga) in sheep. The results shows E. ruminantium infection was prevalent and widely but unevenly distributed throughout the eight districts under study. These findings should be taken into consideration when future disease control and livestock upgrading programs are implemented.     

In vitro lymphocyte transformation as a herd survey method for bovine paratuberculosis.

The lymphocyte-transformation (LT) test was evaluated for its potential application as a field test for bovine paratuberculosis. Using a whole blood technique, samples from 3 consecutive collection periods were subjected to 3 mycobacterial antigens and to phytohemagglutinin. The results obtained from LT were compared with conventional serologic and cultural methods. A positive LT response to johnin purified-protein derivative (PPD) or avian PPD (or both) was noted in 40% to 60% of the animals tested. The complement-fixation test yielded 4% to 6.7% positive results, the immunodiffusion test between 1.2% and 1.4%, and the direct fecal culture between 2.4% and 6%. The mean of the stimulation indices of all positively responding animals was highest with johnin PPD. Specific stimulation to mammalian PPD occurred between 2.4% and 6% of the animals. The efficacy of the LT test for determining the incidence of infection with Mycobacterium paratuberculosis is discussed.     

Characterization of the long-term immune response to vaccination against Mycobacterium avium subsp. paratuberculosis in Danish dairy cows

Vaccination of cattle against Mycobacterium avium subsp. paratuberculosis (MAP) provides partial protection by delayed shedding of MAP and reduced numbers of clinically affected animals. The duration of vaccine induced immune response is not known. The primary objective of this study was therefore to characterize the long-term effect of whole-cell based vaccination against MAP on the immune response. A secondary objective was to evaluate whether immunodiagnosis of MAP and Mycobacterium bovis infections is affected by MAP vaccination. Two studies were performed: (1) A retrospective longitudinal study including 895 vaccinated and 2526 non-vaccinated dairy cows in 9 Danish dairy herds aiming at characterizing the long-term antibody-response to vaccination; and (2) a cross-sectional study of responses in the IFN-γ assay carried out in 140 vaccinated animals in two herds to evaluate the effect of vaccination on the cell-mediated immune response and to evaluate a possible interference with the diagnosis of M. bovis infections. The results showed that 37% of samples from vaccinated animals and 5% of samples from non-vaccinated animals, respectively, were test positive in the milk antibody ELISA. The prevalence of antibody responses of the vaccinated animals was relatively constant from 2 to 6 years of age, but decreased in older animals. Among the 140 vaccinated animals 88% tested positive with the IFN-γ test to johnin PPD and 50% responded to PPDb with IFN-γ production above a similar cut-off. Although Denmark is free of M. bovis, two of the vaccinated animals responded with higher IFN-γ levels when cultured with PPDb compared to PPDa. In conclusion, immunization with whole-cell MAP vaccines elicits both humoral and cell-mediated immune reactions, which may interfere with surveillance and diagnosis of both MAP and M. bovis infections using currently available tests.     

Gastrointestinal nematode infections in small ruminants under the traditional husbandry system during the dry season in southern Ethiopia

A cross-sectional study was conducted from November 2008 to February 2009 to investigate the prevalence and intensity of infection and risk factors of gastrointestinal (GI) nematodes in small ruminants kept under the traditional husbandry system in two districts in southern Ethiopia. Faecal samples collected from a total of 510 small ruminants (284 sheep and 226 goats) and analysed by a modified McMaster technique revealed that 222 animals (43.5%) were found to be infected with one or more GI nematodes. Five identical genera of nematodes were found in both sheep and goats, which in order of predominance were Haemonchus (56.3%), Trichostrongylus (39.6%), Oesophagostomum (22.9%), Trichuris (21.6%) and Bunostomum (10.4%). No significant (p > 0.05) differences were observed between sheep and goats proportions except for Trichuris (p < 0.05). In both sheep and goats, most of the animals were heavily infected showing faecal egg counts (FECs) above 1,200 epg. Sheep had a significantly (p < 0.05) higher mean FEC than goats. In sheep and goats, both the prevalence of GI nematodes and mean FEC were significantly (p < 0.001) associated with body condition score and faecal consistency but not with district, sex and age (p > 0.05 for each factor). In conclusion, the observation of a strong association of GI nematodes with poor body condition coupled with heavy intensity of infection in the majority of infected animals and an abundance of nematode genera of widespread economic and pathological significance warrants the institution of appropriate control measures that should necessarily include improvement of the nutritional status of the animals.     

Lymphoproliferative responses in pigs infected with Mycobacterium avium

Naturally infected cases of swine mycobacteriosis were divided into two groups, localized infection (LI) and disseminated infection (DI). Lymphoproliferative response (LPR) was then examined to estimate their immunological states. Both control and LI groups showed strong response to Concanavalin A (Con A) and phytohemagglutinin (PHA) in the LPR, and lymphocytes recovered from the LI responded well to purified protein derived from M. avium (PPD). On the other hand, the DI group showed weak response to both Con A and PHA, despite their strong response to PPD stimulation. These data suggest that the low LPR to Con A and PHA observed in the DI groups was probably not due to the general unresponsiveness of T-cells.     

Sequential development of histologic lesions and their relationship with bacterial isolation, fecal shedding, and immune responses during progressive stages of experimental infection of lambs with Mycobacterium avium subsp. paratuberculosis

Understanding pathogenesis during progressive stages of infection by Mycobacterium avium subsp. paratuberculosis (MAP) and finding suitable methods for its diagnosis are key to the control of Johne's disease in animals. Paratuberculosis was experimentally produced in 20 crossbred lambs by oral administration of MAP to study the sequential development of lesions between 10 and 330 days postinfection and to assess commonly used diagnostic methods such as bacterial culture, lymphocyte stimulation test (LST), and enzyme-linked immunosorbent assay (ELISA) during progressive stages of infection. Histologic lesions were classified into four grades from grade 1 (least severe) to grade 4 (most severe) on the basis of location of granulomatous lesions in different regions and layers of intestines, their association with intestinal lymphoid tissues, pattern and distribution of lesions, types of cellular infiltration, and presence of acid-fast bacilli. It is evident that infection first establishes in lymphoid tissues of the small intestine, possibly at multiple sites, producing segmental lesions and from there spreads to lamina propria and local lymph nodes. Wide variability in the histologic lesions in relation to postinfection periods and initial tropism of MAP to the intestinal lymphoid tissues (Peyer's patches) suggests a differential susceptibility of young animals, possibly because of compositional phenotypic variation of Peyer's patches influencing subsequent course of infection. Histopathology was found to be a better indicator of paratuberculous infection than bacteriology in sheep. The LST (reflecting the cellular immune response) and ELISA (reflecting the humoral immune response) had overall sensitivities of 65% (11 of 17) and 42% (8 of 19), respectively, in sheep with different types of pathology but when employed together could detect about 88% of infected animals.     

Differences in the peripheral immune response between lambs and adult ewes experimentally infected with Mycobacterium avium subspecies paratuberculosis

The peripheral immune response, and its relationship with the outcome of the infection according to the age of the animal, has been investigated in young lambs and adult ewes experimentally infected with two different doses of Mycobacterium avium subspecies paratuberculosis (Map). Sixteen 1.5-month-old lambs out of 24 and 23 adult ewes out of 30 were orally challenged with an ovine Map field isolate. Animals were divided into two groups: HD, infected with a higher dose of Map and LD, with a lower dose. The remaining animals were used as uninfected control groups. Animals were euthanized at 110-120 and 210-220 days post-infection (dpi). Along the experiment, the humoral response and the specific and non-specific IFN-γ production were assessed. An intradermal skin test (IDT), using avian PPD, was also performed at 90 and 195 dpi. Samples of intestine and related lymphoid tissue were taken for histological, bacteriological and PCR studies. The Ab and IFN-γ production as well as the IDT response appeared earlier and with more intensity in the adult ewes compared to the lambs. The basal non-specific IFN-γ levels increased only in the adult ewes from the HD group. Animals from the LD and HD groups were positive to PCR; however, lesions consistent with paratuberculosis were exclusively observed in the HD group, both in lambs and in adult sheep, but they only progressed to more advanced stages in the former. These results suggest that the peripheral immune response induced by Map infection in the adult ewes is more efficient to control the progression of the infection than in lambs. This could likely be due to the existence of previous contacts with Map or other mycobacteria in the adult sheep compared to the young lambs.     

Molecular survey of <Emphasis Type="Italic">Toxoplasma</Emphasis> infection in sheep and goat from Fars province,Southern Iran

Toxoplasmosis is a widespread zoonotic disease that causes significant morbidity and mortality in human fetus and in immunocompromised patients. Moreover, it becomes a major cause of abortion in sheep and goats. Since consumption of meat of infected lamb and goat is considered as the main sources of human infection in Iran, this study was undertaken to determine the prevalence of Toxoplasma infection in edible tissues of sheep and goats in Shiraz in 2008. Samples of brain, tongue, liver, and muscles of neck, intercostals, and femoral were taken from 56 sheep and 22 goats and tested by PCR. The total prevalence of Toxoplasma infection among animals was found to be 33.3%. Five out of 22 goats (22.7%) and 21 out of 56 sheep (37.5%) were infected by Toxoplasma. Differences between the prevalence rate of infection among females (nine out of 14 = 46%) and males animals (12 out of 45 = 29.5%) was significant (P = 0.013). Furthermore, a positive correlation was found between the age of animals and the rate of infection; animals greater than 2 years old showed a higher rate of infection (47%) in comparison with those less than 2 years old (25%, P = 0.04). The highest infected tissue was tongue (21.8%) followed by brain (19.2%) and femoral and intercostal muscles (17.9%). This study demonstrated a high level of Toxoplasma infection in slaughtered animals in Shiraz and these should be considered as the main sources of infection for human population in the region.     

Bovine paratuberculosis I. A herd study using complement fixation and intradermal tests.

A dairy herd (102 cattle) which had been enrolled under a paratuberculosis control program for two years utilizing a complement fixation test (carbohydrate antigen) and intradermal skin test (johnin PPD) was subjected to two further herd tests and followed to slaughter to determine infection status by culture and histology. Mycobacterium paratuberculosis infection was demonstrated in 37 of the animals of which only five were considered reactors on the basis of the last two herd tests applied. Cultural and histopathological evaluation indicated the testing procedures had eliminated heavily infected animals. The limitations of these testing procedures under free stall housing conditions are discussed.     

Application of different methods for the diagnosis of experimental paratuberculosis in goats

The diagnosis of subclinical paratuberculosis is still considered a major problem worldwide. As part of investigating diagnostic strategies for the paratuberculosis infection, sequential results of various diagnostic methods in a progressive experimental infection in goats were evaluated. Twenty-three goat kids were divided into three groups: the infected, contact and control, comprising 10, five and eight goats respectively. Animals of the infected group were orally inoculated on seven occasions with 5 ml of inoculum containing 2 x 10(9)Mycobacterium avium ssp. paratuberculosis per ml. Lymphoycte proliferation test using johnin PPD detected paratuberculosis infection from 60 days post-infection (DPI) onwards. The johnin PPD was found to be a better antigen for the proliferative assays as compared with the sonicated antigen. The faecal smear examination with acid-fast staining detected more goats as positive than bacterial culture and polymerase chain reaction (PCR). Lipoarabinomannan enzyme-linked immunosorbent assay (ELISA) started detecting infected goats from 150 DPI onwards followed by indirect ELISA and agar gel immunodiffusion from 180 DPI onwards. Histological examination was confirmatory and detected five infected goats as positive.     

A longitudinal study to evaluate the diagnostic potential of a direct faecal quantitative PCR test for Johne's disease in sheep

A longitudinal study was carried out to evaluate the diagnostic potential of the previously developed direct faecal real-time quantitative PCR (QPCR) assay (Kawaji et al., 2007) for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infected sheep. Of the 58 sheep, 38 were orally inoculated with MAP, while 20 controls were maintained separately from the infected group throughout the trial. All animals were tested by QPCR, faecal culture and serum ELISA pre-inoculation and at 4, 8 and 13 months post-inoculation, and were necropsied at 13 months post-inoculation. Eighteen out of 38 inoculated sheep were detected by QPCR to be shedding MAP in faeces at 4 months post-inoculation, while only one sheep was positive in faecal culture at this time point. At 8 months post-inoculation, MAP DNA was detected in faeces of all inoculated sheep by QPCR, while MAP organisms were isolated from only 34% of the inoculated animals by faecal culture. The QPCR results for faecal samples that were collected at necropsy demonstrated that faecal QPCR was more sensitive than culture of intestinal tissues for MAP. The QPCR assay was confirmed to be a sensitive and specific ante-mortem diagnostic test for MAP in sheep, circumventing faecal culture which is a less sensitive and highly time consuming test. Quantification of MAP DNA in faeces by QPCR may provide immediate information to estimate the stage of the infection as well as the risk of transmission from infected animals.     

Effect of Trypanosoma vivax infection on body temperature, feed intake, and metabolic rate of West African dwarf goats.

Thirty-two mature dwarf goats weighing between 16 and 30 kg (22.7 +/- 3.7, SD) were used to study the effect of Trypanosoma vivax infection on rectal temperature (RT), feed intake (DMI), and metabolic rate. Sixteen of the goats were infected intravenously with 14 X 10(6) T. vivax each; the 16 others served as controls. Animals were fed at about 1.1 times maintenance. Heat production was measured from 1 wk preinfection to 6 wk postinfection. From data on successive 9-min periods, heat production was calculated per 24-h period and separately for 0700 to 2000 (day period) and for 2000 to 0700 (night period). Rectal temperature was measured twice weekly. Compared with controls, animals infected with T. vivax developed and maintained a 1 degree C higher RT and a higher metabolic rate. After the prepatent period of 5 to 7 d, during which RT remained normal, all infected goats had a period of about 7 d with constant high temperatures. After that initial episode, RT fluctuated. Heat production of infected animals was increased by 15.6 kcal.d-1.kg-.75, or about 16%. This increase in heat production was greater during the night (22 kcal.d-1.kg-.75) than during the day (14 kcal.d-1.kg-.75). After T. vivax infection, large differences in DMI among animals were apparent. In four animals, a clear relation between DMI and RT was noted, but in 12 animals no such relationship was apparent.     

Prevalence of ovine and caprine oestrosis in Ambo,Ethiopia

A study was carried out to estimate the prevalence, larval burden and risk factors of ovine and caprine oestrosis from December 2007 to May 2008 on 554 heads of randomly selected sheep and goat slaughtered at Ambo town, Western Shoa, Ethiopia. The results show an overall prevalence of 59.9% with infection rate of 69.8% and 47.3% in sheep and goats respectively. No statistically significant difference in the prevalence was noted with regard to the assumed risk factors like sex, nose color, face color, horned versus polled, origin, and months (p > 0.05). Sheep were nearly twice more likely to be infected as compared to goats (p = 0.0001, odds ratio (OR) = 1.975). Age of the animals was found to be protective (OR = 0.579; 95% confidence interval = 0.393, 0.853; p = 0.006). As compared to very fat animals, poor (p = 0.040, OR = 4.834), medium (p = 0.049, OR = 4.198), and fat (p = 0.022, OR = 5.795) body condition animals are more likely to be infected by Oestrus ovis larvae. Nasal and sinus cavity pathology is positively correlated with the total larval count (r = 0.56, p < 0.0001). Out of a total of 3,770 larvae collected, 57.5% were L1, 30.8% L2, and 11.7% L3 larvae. All the three larval instars were seen throughout the study months. It is concluded that oestrosis is a common problem in the study area and more prevalent in sheep than goats, in adult than young, and in animals with poor body condition.     

Distribution pattern of bovine viral diarrhoea virus type 1 genome in lymphoid tissues of experimentally infected sheep

In this study, cellular localization and the distribution pattern of BVDV genome in lymphoid tissues during the course of experimental acute BVDV-1 infection of sheep was investigated. Tonsils, mesenteric lymph nodes (MLN) and spleen were collected on 3, 6, 9, 12 and 15 days post infection (dpi) from twenty 4-month-old lambs, experimentally inoculated intra-nasally with 5?×?10⁵ TCID₅₀ of a non-cytopathic (ncp) BVDV-1 isolate, Ind-17555. Tissues collected from ten mock-infected lambs served as controls. In situ hybridization (ISH) was carried out in paraformaldehyde fixed paraffin embedded tissue sections using digoxigenin labelled riboprobe targeting 5′-UTR of BVDV-1. BVDV genome was detected at all the intervals from 3 dpi to 15 dpi in the lymphoid tissues with variations between the intervals and also amongst the infected sheep. During the early phase of acute infection, presence of viral genome was more in tonsils than MLN and spleen, whereas the distribution was higher in MLN during later stages. BVDV-1 genome positive cells included lymphocytes, macrophages, plasma cells, reticular cells and sometimes crypt epithelial cells. Genome distribution was frequently observed in the lymphoid follicles of tonsils, MLN and spleen, besides the crypt epithelium in tonsils, paracortex and medullary sinus and cords of MLN. Most abundant and widespread distribution of BVDV-1 genome was observed on 6 dpi while there was a reduction in number and intensity of positive signals by 15 dpi in most of the infected animals. This is the first attempt made to study the localisation of BVDV-1 in lymphoid tissues of acutely infected sheep by in situ hybridization. The results show that the kinetics of BVDV-1 distribution in lymphoid tissues of experimentally infected non-pregnant sheep follows almost a similar pattern to that demonstrated in BVDV infected cattle.