Canadian regulatory requirements for recombinant fish vaccines.

In Canada, veterinary biological products derived by using conventional and new techniques of biotechnology are licensed and regulated under the Health of Animals Act and Regulations. Biological products include vaccines, bacterins, bacterin-toxoids and diagnostic kits which are used for the prevention, treatment or diagnosis of infectious diseases in all species of animals, including fish. Veterinary biologicals are licensed on the basis of fulfillment of four criteria: purity, potency, safety and efficacy. A risk-based approach is used to evaluate the safety of the product in target species, as well as non-target species, humans and the environment. On the basis of biological characteristics, biotechnology derived veterinary biologicals have been divided into two broad categories, high and low risk products. The paper describes the regulatory framework for the licensing of veterinary biologicals in Canada, with emphasis on the regulatory considerations for recombinant fish vaccines. Stages of movement of the product from research in a contained laboratory facility to a fully licensed product for free sale are discussed. The requirements for field testing and environmental assessment involved in these stages are highlighted. Manufacturers and researchers who intend to commercialize experimental vaccines are encouraged to consult with the Veterinary Biologics and Biotechnology Section early in the product development process so that the research data and quality assurance documentation are consistent with regulatory requirements.

Serum haptoglobin as an indicator of the acute phase response in bovine respiratory disease.

The early stages of the host response to infectious agents include a number of physiologic changes, collectively known as the acute phase response. The acute phase response is comprised of reactions localized at the site of infection, as well as the initiation of systemic responses, which include a rapid increase in the serum concentration of some proteins, known as acute phase proteins (APP). Using polyacrylamide gel electrophoresis, we detected two APP of approximately 22 and 37 kDa molecular weight in sera obtained from cattle with bovine respiratory disease (BRD). Based on their presence in the sera of sick, but not normal animals, the molecular weights, N-terminal amino acid sequence analysis, and the ability to bind hemoglobin, we identified these proteins as the alpha and beta subunits of haptoglobin. The haptoglobin molecule and the alpha subunit were isolated from serum, purified, and used to produce monoclonal and polyclonal antibodies. With these reagents, an enzyme linked immunosorbent assay was developed to measure the concentration of haptoglobin in bovine serum. Using an experimental model of BRD induced by a sequential challenge of calves with bovine herpesvirus type-1 and Pasteurella haemolytica, we observed a temporal relationship between the increase in haptoglobin concentration in serum and the onset of bacterial infection. The haptoglobin concentration ranged from undetectable in the serum of most calves prior to challenge, to greater than 1 mg ml(-1) in over one-third of the calves at the height of disease. Furthermore, the concentration of haptoglobin was associated significantly with other measures of the severity of disease. Together, these results indicate that quantification of acute phase proteins in animals with BRD could be a valuable diagnostic and prognostic aid.

Fragment Bb of bovine complement factor B: stimulatory effect on the microbicidal activity of bovine monocytes.

We have previously shown that the Bb fragment of bovine complement factor B activates bovine monocytes and neutrophils. The activation was demonstrated by the enhanced uptake of 3H-deoxyglucose. To investigate the potential effect of fragment Bb on the microbicidal activity of bovine monocytes, a direct method was used. This method involves an initial ingestion period at 37 degrees C followed by repeated washing. The decrease in the total number of viable intracellular Staphylococcus aureus during the reincubation of the bacteria with bovine monocytes determines the intracellular killing. Maximal intracellular killing was seen when the monocytes containing the ingested S. aureus was incubated with fresh bovine serum (mean +/- SEM = 73.4 +/- 1.4%). On incubation of the monocytes, containing the ingested bacteria with heat-inactivated bovine serum, 32.5 +/- 0.7% of the intracellular bacteria were killed. When affinity-purified bovine factor Bb was added to the heat-inactivated serum, the intracellular killing capacity was almost restored (65.8 +/- 1.5%). When monocytes were incubated with medium alone, they killed 22.4% of the intracellular microorganisms. When fragment Bb (25 micrograms/mL) was added to the medium, the intracellular killing of S. aureus doubled (46 +/- 1.29%). We conclude that the Bb fragment of bovine complement factor B stimulates bovine monocytes in their microbicidal activity.

Bb fragment of bovine complement factor B: stimulation of the oxidative burst in bovine monocytes.

In a previous study, we reported that fragment Bb of bovine complement factor B activated bovine monocytes, as demonstrated by the uptake of 3H-deoxyglucose. In the present study, the effects of Bb on the production of superoxide anion and hydrogen peroxide by bovine monocytes was investigated. The production of superoxide was measured by the superoxide dismutase inhibitable reduction of cytochrome c. The change in absorbance was determined by a spectrophotometer at a wavelength of 550 nm. Hydrogen peroxide production was measured by the horse-radish peroxidase-dependent oxidation of phenol red. The resulting color change was measured by a spectrophotometer at a wavelength of 620 nm. Fragment Bb (20 micrograms/mL) induced the generation of 0.96 +/- 0.2 (mean +/- SEM) nanomoles of superoxide/2.5 x 10(5) monocytes at 5 min. The production of superoxide peaked at 15 min (1.24 +/- 0.3 nanomoles). The production of hydrogen peroxide was also rapid: 0.195 +/- 0.05 nanomoles/2.5 x 10(5) monocytes at 5 min with a peak at 15 min (0.250 +/- 0.04 nanomoles). These observations show that fragment Bb, which has serine protease activity, induces bovine monocytes to generate reactive oxygen intermediates such as superoxide and hydrogen peroxide.

Activation of bovine monocytes and neutrophils by the Bb fragment of complement factor B: demonstration by the uptake of 3H-deoxyglucose.

The Bb fragment is the enzymatically active split product of bovine complement factor B. The Bb fragment was obtained after zymosan treatment of fresh bovine serum and fractionation of the treated serum, first over diethylaminoethyl-Sephacel and then over an affinity column made up of monoclonal antibody to bovine Bb, coupled to cyanogen-bromide-activated Sepharose. Purified Bb has a molecular weight of 64,000, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The ability of purified Bb to activate phagocytes was assessed. The activation assay was based on the principle that the primary source of energy for the phagocytes is obtained from glucose. 3H-deoxyglucose, a nonmetabolizable analogue of glucose, was used to obtain the quantitative measurement of the activation process. The activation by Bb was shown by the uptake of the labelled deoxyglucose in the phagocytic cells and was comparable to the activation caused by phorbol myristate acetate and N-formyl-L-methionyl-L-leucyl-L-phenylalanine, run in parallel. These data showed that fragment Bb activates bovine monocytes and neutrophils and also suggested that, when generated after complement activation, Bb may stimulate monocytes and neutrophils for enhanced phagocytosis.

A study on the reservoir status of Q-fever in avifauna, wild mammals and poikilotherms in Uttar Pradesh (India).

Fifteen species of free-living birds (pigeon, mynah, house-sparrow, crow, vulture, owl, swallow, parrot, heron, duck, guinea fowl, hawk, kite, dove and peacock), 11 species of small/large wild mammals (rat, bandicoot, house mouse, shrew, bat, mongoose, ant eater, Jackal wild cat, chinkara and tiger) and 5 species of poikilotherms (snakes, python, tortoise, monitor and eel) were screened for evidence of Q-fever infection by the capillary agglutination test on sera to detect antibodies and/or by attempts to demonstrate Coxiella burnetii in samples of visceral organs. Sero-reactors were observed among mynah (19/69), owlet (1/6), pigeon (1/11), swallow (6/200), parrot (13/56), rat (3/21), shrew (1/24), bat (2/14) and snake (7/23). Eleven strains of the organism comprising 3 from mynah, 2 from rats, and 1 each from parrot, crow, swallow, bandicoot, chinkara and python were isolated. This appears to be the first record of natural Q-fever infection in free-living birds in India. C. burnetii was also recovered in 1 of 12 water samples processed from ponds. Possibility of contamination of ponds with C. burnetii from infected water-fauna has been discussed.

The occurrence of salmonellae in zoo animals in Uttar Pradesh and Delhi (India).

The occurrence of salmonellae in a variety of zoo animals including carnivores, primates, ruminants, avifauna, rodents etc. was investigated. Of 783 samples collected from Lucknow, Delhi and Kanpur (India) Zoological Parks, only 5 yielded Salmonella serotypes. These included S. typhimurium, from a leopard and a wild cat, S. enteritidis from two black and white rats and a strain of Salmonella group E1 from a leopard.

The occurrence of salmonellae in free-flying-avifauna: isolation and antibiogram.

The occurrence of salmonellae in a variety of free-flying birds was investigated. Of 790 intestinal-content-samples examined, 20 yielded different Salmonella serotypes, which included 10 strains of S. saint-paul, 4 of S. bareilly, 3 of S. weltevreden, 2 of S. typhimurium and 1 of Salmonella E1 group. Common Mynah, house-sparrow, swallow, grey-partridge, parrot and crow were found positive for the presence of salmonellae. Antibiogram of the isolates was studied against 14 common chemotherapeutic agents.

The occurrence of salmonellae in rodent, shrew, cockroach and ant.

The occurrence of salmonellae in common house-pests viz. rats, house-mice, shrews, cockroaches and ants was investigated during the period of Jan. 1976 to July 1979. Of 767 samples examined, 43 yielded different Salmonella serotypes. The isolation of salmonellae was made from 16 of 254 rats, 11 of 109 house-mice, 11 of 104 shrews, 3 of 270 cockroaches and 2 of 30 ants. The different Salmonella serotypes isolated included S. saint-paul, S. bareilly, S. newport, S. weltevreden, S. enteritidis, S. typhimurium, S. hvittingfoss, S. anatum, S. metopeni, S. waycross and S. paratyphi B. One shrew yielded three different serotypes of salmonellae, while dual infection was detected in three shrews and one rat.

Sero-epidemiological studies on coxiellosis in animals and man in the state of Uttar Pradesh and Delhi (India).

The prevalence of antibodies to Coxiella burnetii, the causative agent of Q-fever, was studied in domestic animals either at the farm or at the slaughter house and man in Delhi and Uttar Pradesh (U.P.). Evidence of Q-fever infection was observed in 24.29% of 490 cattle, 16.79% of 536 sheep, 16.02% of 1011 buffaloes, 15.85% of 1937 goats, 14.67% of 184 pigs and 14.29% of 49 street dogs. Twenty percent of 55 dairy cows, 9.52% of 21 dairy goats and 5.71% of 35 dairy buffaloes were found positive for C. burnetii antibodies in their milk. Of 1636 human sera samples tested, 249 (15.22%) were positive. Human sero-positive reactors in U.P. and Delhi were 15.59% and 14.39% respectively. Factors of age, sex, season antibody titres, co-existance of Q-fever and brucellosis and public health significance of high Q-fever infection in meat and dairy animals under the prevalent socio-economic conditions are discussed.

Sero-epidemiologic investigations on brucellosis in the states of Uttar Pradesh (U.P.) and Delhi (India).

Sero-prevalence of brucellosis in man and animals was studied during the years 1976 and 1977. Samples were collected from Hospitals/slaughter houses/livestock farms located in Delhi and different districts of Uttar Pradesh (U.P.). The sera samples tested were from 1685 men, 1607 goats, 438 sheep, 244 pigs, 361 cattle, 551 buffalos, 50 dogs, 318 equine and 43 free living animals. The percentage of seropositivity, excluding doubtful ones, was recorded as: man 0.89, goat 5.53, sheep 3.42, pigs 15.98, cattle 6.37 buffalo 4.9 and equine 12.89. Additionally an evidence of agglutinins was also detected in a python serum sample. It was observed that occupation, age, sex and season had a bearing on the prevalence of the disease.

Toads as reservoirs of salmonellae: prevalence and antibiogram.

Of 570 intestinal-content-samples of toads examined, 40 yielded salmonellae, which included 26 strains of S. goverdhan, 9 of S. bareilly, 2 of S. richmond, and 1 each of S. typhimurium, S. weltevreden and S. newport. From 52 visceral-organ samples, 6 strains of S. goverdhan and 1 of S. typhimurium were isolated. Antibiotic sensitivity studied against 18 chemotherapeutic agents, revealed that although the isolates were sensitive to most of the antibiotics, all of them were resistant to bacitracin, novobiocin and oleandomycin. To sulfonamides, erythromycin and pencillin, respectively, 42, 8 and 3 isolates were resistant.

The occurence of coxiellosis among rodents and shrews in the Tarai area of Uttar Praedesh.

Rodents and shrews were screened for serologic evidence of Coxiella burnetii. Attempts were made to isolate the organism from the spleen and liver. Seroreactors: total positive/total tested (% positive), in rats (Rattus rattus, R. norvegicus), ground shrews (Suncus murinus), bandicoots (Bandicota indica, B. bengalensis) and the house mouse (Mus musculus), respectively, were 13/105 (12.38), 6/42 (14.3), 2/15 (13.3) and 1/7 (14.3). Of the eight rickettsial isolants recovered including four from field and household rats, three from ground shrew and one from bandicoots, only three comprising one each from rat, shrew and bandicoot, could be typed as C. burnetii. This appears to be the first record of rodents and an insectivore as reservoirs of C. burnetii in India.

Poikilotherms as reservoirs of Q-fever (Coxiella burnetii) in Uttar Pradesh.

Water snakes (Natrix natrix), rat snakes (Ptyas korros), cobras (Naja naja), pythons (Python molurus), tortoises (Kachuga sp.), plankton fish (Cirrhina mrigala), frogs (Rana tigrina), toads (Bufo sp.) and monitors (Varanus indicus) were screened for evidence of Q-fever infection by the capillary agglutination test on sera to detect antibodies and/or by attempts to demonstrate Coxiella burnetii in spleen and liver samples. Sero-reactors were observed among water and rat snakes, pythons and tortoises. The organism was isolated from the spleen and liver of the monitor, tortoise and python.

Experimental infection of Coxiella burnetii in chicken: clinical symptoms, serologic response, and transmission through egg.

Young laying hens were infected with Coxiella burnetii to study egg transmission, clinical and serologic responses, excretion of the agent in feces, and its persistence in internal organs. No clinical symptoms were noticed. The birds developed good titers in a capillary agglutination test by 13 days postinfection (DPI), which peaked at 30 DPI and therafter declined gradually, becoming negative in some birds around 90 DPI. In vivo and in vitro egg transmission of the agent could not be demonstrated. C. burnetii was recovered at 90 DPI from spleen and liver but not from kidney and ovary.

Sero-epidemiology of Q-fever in poultry.

The sero-epidemiology of Q-fever was studied by capillary agglutination test at 25 poultry farms in the Nainital and Ajmer districts of Uttar Pradesh and Rajasthan. Of 589 birds tested, 78 (13.24%) had Q-fever agglutinins (CAT titers 1:8 to 1:64), involving 16 of the farms. There were more sero-reactors in Ajmer (17.56%) than in Nainital (3.35%). The sero-positive reactors were respectively 19.74% and 5.55% among the age groups above and below 6 months. The breed difference and comparatively high infection rates in poultry attendants of a Q-fever-positive farm are discussed.

An outbreak of fowl cholera due to Pasteurella gallinarum in Uttar Pradesh (India).

An acute outbreak of fowl cholera in one-month-old chickens was investigated. Pasteurella gallinarum was isolated in pure culture from the heart blood of two moribund chicks. One of the isolants on experimental inoculation was found to be nonpathogenic for rabbits, mice, and chickens. It did not provide protection in rabbits against a virulent strain of P. multocida. This seems to be the first record of the isolation of P. gallinarum in India.

Morphological differences in longissimus dorsi muscle of pig, buffalo, sheep, goat and poultry.

With a view to establish morphological differences between the muscles of pig, buffalo, sheep, goat and poultry, longissimus dorsi were examined. The average fibre cross-sectional area, average fibre diameter, number of fibres per unit area, fascicle size and number of fascicles per unit area have been studied. The size of the muscle fibres showed a decrease in the following order: pig, buffalo, sheep, goat and poultry. In poultry the muscle fibres were thinner and the fibre density values were greater in comparison to other species. The fibres were the coarsest in the pig. A slight difference in the fascicle size between the different species could be appreciated, the pig fascicles being generally large.