Molecular Characterization of Foot-and-Mouth Disease Viruses Collected in Tanzania Between 1967 and 2009.

This paper describes the molecular characterization of foot-and-mouth disease viruses (FMDV) recovered from outbreaks in Tanzania that occurred between 1967 and 2009. A total of 44 FMDV isolates, containing representatives of serotypes O, A, SAT 1 and SAT 2 from 13 regions of Tanzania, were selected from the FAO World Reference Laboratory for FMD (WRLFMD) virus collection. VP1 nucleotide sequences were determined for RT-PCR amplicons, and phylogenetic reconstructions were determined by maximum likelihood and neighbour-joining methods. These analyses showed that Tanzanian type O viruses fell into the EAST AFRICA 2 (EA-2) topotype, type A viruses fell into the AFRICA topotype (genotype I), type SAT 1 viruses into topotype I and type SAT 2 viruses into topotype IV. Taken together, these findings reveal that serotypes O, A, SAT 1 and SAT 2 that caused FMD outbreaks in Tanzania were genetically related to lineages and topotypes occurring in the East African region. The close genetic relationship of viruses in Tanzania to those from other countries suggests that animal movements can contribute to virus dispersal in sub-Saharan Africa. This is the first molecular description of viruses circulating in Tanzania and highlights the need for further sampling of representative viruses from the region so as to elucidate the complex epidemiology of FMD in Tanzania and sub-Saharan Africa.

Protective antibody response following oral vaccination of feral pigeons (Columba livia) with Newcastle disease vaccine (strain I-2) coated on oiled rice.

The novel vaccination technique for feral pigeons was developed in the present study. Multi-age feral pigeons were vaccinated orally with Newcastle disease (ND) strain I-2 vaccine coated on oiled rice. The results showed that 14 days after vaccination 40% of pigeons seroconverted with HI GMT of ≥3 log2 whereas 28 days after vaccination the seroconversion rate of these birds reached 100%. Moreover, all vaccinated pigeons survived the challenge of virulent Newcastle disease virus (NDV). The findings from the present study indicated that the use of ND (strain I-2) vaccine in feral pigeons is feasible and resulted into the production of protective antibody response. Thus ND I-2 vaccine may prevent the spread of NDV to other birds particularly chickens. Furthermore the use of oral vaccine in feral multi-age pigeons overcomes the difficulty of catching these birds for individual vaccination.

Vaccination of chickens using raw rice coated with novel trehalose nano-organogels containing Newcastle disease (strain I-2) vaccine.

The formulation and evaluation of trehalose nano-organogels for storage and oral delivery of Newcastle disease (ND) strain I-2 vaccine to chickens were carried out in this study. Trehalose sugar was blended with vegetable oil to form nano-organogels where trehalose also acted as a stabilizer against thermal inactivation of I-2 ND virus. Results from infectivity titration assay indicated that the titre of 10(7.5) EID(50)/0.1 mL was maintained after 12 weeks of storage of nano-organogel I-2 vaccine at ambient room temperature. Serology results showed that 33% chickens which were vaccinated with nano-organogel I-2 vaccine after 14 days had HI antibody titres of > or = 3.0 log(2) with GMT of 2.3. Moreover, results showed 100% of chickens vaccinated with nano-organogel I-2 vaccine had the mean antibody titres of 3.4 and 3.7 log(2) at 21 and 28 days after vaccination, respectively. All vaccinated chickens (100%) survived the challenge of virulent ND virus whereas all unvaccinated chickens succumbed to challenge and died of signs consistent with ND. The findings from this study showed that the nano-organogel I-2 vaccine was stable at room temperature, safe and produced protective antibody response in vaccinated chickens. Moreover the nano-organogel I-2 vaccine was used for oral administration and hence is suitable for mass vaccination. However, optimization of the formulation of trehalose nano-organogel vaccine is required in order to achieve its application potentials.

Oral vaccination of chickens against Newcastle disease with I-2 vaccine coated on oiled rice.

Antibody response produced by Newcastle disease virus (NDV, strain I-2) when given orally through oiled rice to chickens was determined. Serum samples were collected before and at a weekly interval for 28 days after vaccination and tested for haemagglutination inhibition (HI) antibody to NDV. The results showed 7 days after vaccination HI antibody titre log(2) was 3.8. Moreover, 14 and 28 days after vaccination HI antibody titre log(2) reached 6.5 and 8.0, respectively. All unvaccinated chickens were negative to NDV antibody throughout the study. Significant finding from the present study is that 7 days after vaccination chickens had produced protective antibody against NDV; this is in contrast to previous studies. Therefore, I-2 vaccine coated on the oiled rice is efficacious as it protects chickens from challenge with NDV.

Protective antibody response produced by the chickens vaccinated with green coloured thermostable Newcastle disease virus.

The efficacy of green-coloured (GC) I-2 Newcastle disease vaccine was determined in the present study. I-2 vaccine was mixed with a green coloured dye and stored at 4 degrees C for 6 months while assayed for the virus infectivity at a monthly interval. Chickens were vaccinated with the GC vaccine by eye drop. Serum samples were collected from all birds before and after vaccination at weekly interval for 4 weeks and tested for haemagglutination-inhibition (HI) antibody against Newcastle disease virus (NDV). These chickens were challenged with NDV virulent strain four weeks after vaccination. The results showed that there was no difference between the infectivity titres of GC and uncoloured vaccines. However, chickens vaccinated with GC vaccine produced higher HI antibody titres than chickens vaccinated with uncoloured vaccine. Results from the challenge trial showed that all vaccinated chickens survived whereas all unvaccinated chickens died. The findings from this study have shown that the GC vaccine is safe and produced protective antibodies against NDV in vaccinated chickens.

Molecular characterization of infectious bursal disease virus (IBDV): diversity of very virulent IBDV in Tanzania.

Nucleotide sequences of the VP2 hypervariable region (VP2-HVR) of 14 infectious bursal disease viruses (IBDVs) isolated in Tanzania from 2001 to 2004 were determined. Phylogenetic analysis showed that the isolates diverged into two genotypes and belonged to the very virulent (VV) type. In the phylogenetic tree, strains in one genotype clustered in a distinct group and were closely related to some strains isolated in western Africa, with nucleotide similarities of 96.1-96.8%, while strains in another genotype were clustered within the European/Asian VV type with nucleotide similarities ranging from 97.5 to 99.3%. Both genotypes were widely distributed throughout Tanzania, and had conserved putative virulence marker amino acids (aa) at positions 222(A), 242(I), 256(I), 294(I) and 299(S). Our findings demonstrate for the first time the existence of both African and European/Asian VV-IBDV variants in Tanzania.

Deduced amino acid sequences surrounding the fusion glycoprotein cleavage site and of the carboxyl-terminus of haemagglutinin-neuraminidase protein of the avirulent thermostable vaccine strain I-2 of Newcastle disease virus.

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F(0) cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain I-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain I-2 had a sequence motif of (112) RKQGRLIG(119), consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain I-2 had a 7-amino-acid extension (VEILKDGVREARSSR. This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain I-2 confirmed its avirulent nature and its Australian origin.

Development of a cell culture method for quantal assay of strain I-2 of Newcastle disease virus.

Repeated titrations of strains of Newcastle disease virus (NDV) are more conveniently undertaken in cell cultures rather than in embryonated eggs. This is relatively easy with mesogenic and velogenic strains that are cytopathic to various cell lines, but is difficult with avirulent Australian isolates that are poorly cytopathic. Strain V4 for example has been shown to be pathogenic iin vitro only to of chicken embryo liver cells. Strain I-2 was reported to produce cytopathic effect (CPE) on chicken embryo kidney (CEK) cells. The present studies confirmed this observation and developed a quantal assay. CEK cells infected with strain I-2 developed CPE characterized by degeneration, rounding, granularity and vacuolation, and the formation of synctia. End points were readily established by microscopic examination of fixed and stained cells. In virus infectivity studies on strain I-2, where multiple titrations are required and where large numbers of samples are used, titration using CEK cell grown in microtitre plates is recommended. Such studies may not be feasible in embryonated eggs.

Determination of organ tropism of Newcastle disease virus (strain I-2) by virus isolation and reverse transcription-polymerase chain reaction.

The vaccines I-2 and V4 are avirulent strains of Newcastle disease virus. Organ tropism of strain V4 has been determined and the virus has a predilection for the digestive tract. Tropism of strain I-2 has not yet been determined. The objective of this study was to determine the distribution of strain I-2 in various body organs and fluids following vaccination in comparison with V4. Four-week-old chickens were vaccinated by eye drop separately with these two avirulent strains. Virus isolation and the reverse transcription-polymerase chain reaction technique were employed to detect I-2 and V4 viruses in various tissues and body fluids for 7 days following vaccination. Tissues from the respiratory tract showed earlier positive signals than tissues from other organs for chickens vaccinated with strain I-2. Conversely, tissues from mainly digestive tract produced earlier positive signals than from respiratory tract and other organs from chickens vaccinated with strain V4. In early infection, strain I-2 had preferential predilection for the respiratory tract and strain V4 for the digestive tract. Later after vaccination, other organs showed positive results from chickens vaccinated with both I-2 and V4 strains. The differences in organ tropism observed in this study suggest that I-2 may perform better than V4 as a live vaccine strain.

Development of a rapid biological assay for determination of potency of Newcastle disease vaccine (strain I-2).

A rapid biological assay based on incubation time has been developed for determination of the potency of Newcastle disease virus strain I-2 vaccine. It is based on the observation that the interval between inoculation and the first detection of haemagglutinin (HA) depends on the titre of the vaccine inoculated. Chicken embryonated eggs were inoculated with different titres (10(9), 10(6) and 10(3) EID(50)/0.1 ml) of vaccine and incubated for 24 h. At hourly intervals, 5 eggs from each vaccine titre were tested for the presence of HA. The results showed that the HA activity was detected from 5, 11 and 15 h after inoculation with vaccine doses of 10(9), 10(6) and 10(3) EID50, respectively. On the basis of these results it is suggested that if there is no HA detected from 5 to 11 h after inoculation of eggs with the vaccine virus, the vaccine should not be used to vaccinate chickens as it might have an infectivity titre of less than 106 EID50/0.1 ml, which is equivalent to the recommended single chicken dose. It is concluded that measuring the time between inoculation of the vaccine virus and the onset of HA activity might provide an estimate of the titre of the vaccine within 24 h.

Thermostability profile of Newcastle disease virus (strain I-2) following serial passages without heat selection.

I-2 is an avirulent strain of Newcastle disease virus. During establishment of the I-2 strain master vaccine seed, a series of selection procedures was carried out at 56 degrees C in order to enhance heat resistance. This master seed is used to produce a working seed, which is then employed to produce the vaccine. These two passages are done without further heat selection; however, it is not known how rapidly and to what extent thermostable variants would be lost during further passage. The study was therefore conducted to determine the effect of passage on thermostability of strain I-2. The virus was serially passaged and at various passage levels samples were subjected to heat treatment at 56 degrees C for 120 min. The inactivation rates for infectivity and haemagglutinin (HA) titres were assayed by use of chicken embryonated eggs and HA test, respectively. Thermostability of HA and infectivity of I-2 virus were reduced after 10 and 5 passages, respectively, without heat selection at 56 degrees C. These results suggest that 5 more passages could be carried out between the working seed and vaccine levels without excessive loss of thermostability. This would result in increased vaccine production from a single batch of a working seed.

The control of rinderpest in Tanzania between 1997 and 1998.

In January 1997, Tanzania requested international assistance against rinderpest on the grounds that the virus had probably entered the country from southern Kenya. Over the next few months, a variety of attempts were made to determine the extent of the incursion by searching for serological and clinical evidence of the whereabouts of the virus. At the clinical level, these attempts were hampered by the low virulence of the strain, and at the serological level by the lack of a baseline against which contemporary interpretations could be made. Once it became apparent that neither surveillance tool was likely to produce a rapid result, an infected area was declared on common-sense grounds and emergency vaccination was initiated. The vaccination programme had two objectives, firstly to prevent any further entry across the international border, and secondly to contain and if possible eliminate rinderpest from those districts into which it had already entered. On the few occasions that clinical rinderpest was subsequently found, it was always within this provisional infected area. Emergency vaccination campaigns within the infected area ran from January to the end of March 1997 but were halted by the onset of the long rains. At this time, seromonitoring in two districts showed that viral persistence was still theoretically possible and therefore a second round of emergency vaccination was immediately organized. Further seromonitoring then indicated a large number of villages with population antibody prevalences of over 85%. These populations were considered to have been 'immunosterilized'. Although no clinical disease had been observed in them, it was decided to undertake additional vaccination in a group of districts to the south of the infected area. Serosurveillance indicated that rinderpest could have been present in a number of these districts prior to vaccination. Serosurveillance in 1998 suggested that numerous vaccinated animals had probably moved into districts outside the infected and additional vaccination areas, but did not rule out the continued presence of field infection.

Diagnosis of rinderpest in Tanzania by a rapid chromatographic strip-test.

A simple chromatographic strip-test based on Clearview technology, is under development as a pen-side test for the detection of rinderpest antigen in eye swabs taken from cattle in the field. An outbreak of rinderpest occurred in the northern zone of Tanzania from late February to June 1997. The affected cattle exhibited very mild clinical signs, which made clinical diagnosis difficult. One hundred and seven eye swabs were collected from cattle suspected of infection with rinderpest. These were tested in the field using a prototype of the pen-side test and 13 (12.15%) of the samples were found to be positive for the presence of rinderpest antigen. These were confirmed by ICE. The positive cases were predominantly found in the Ngorongoro district. This demonstrates the usefulness of such a simple, rapid pen-side diagnostic assay, particularly when clinically 'mild' strains of rinderpest are present.

Experimental trials with a thermostable Newcastle disease virus (strain I2) in commercial and village chickens in Tanzania.

Antibody responses in indigenous village and commercial chickens vaccinated with 12 thermostable Newcastle disease (ND) vaccine and protection levels against challenge with a virulent field isolate were determined. The antibody response of village chickens vaccinated by eye drop revealed that 30, 60 and 90 days after primary vaccination, the mean log2 HI titres were 6.1, 5.4 and 3.6, respectively, whereas for commercial chickens, the antibody response after 14, 30 and 90 days were 8.2, 5.1 and 4.2, respectively. Village chickens vaccinated orally via drinking water had mean log2 HI titres of 3.4 after 30 days. After booster vaccination, the mean HI titre was 5.4 and 3.3 after 30 and 60 days post-secondary vaccination (i.e. 60 and 90 days after primary vaccination). Antibody response of mean log2 HI titres of 2.6 was recorded 30 days after primary vaccination orally through food; 30 and 60 days after secondary vaccination (i.e. 60 and 90 days after primary vaccination), mean log2 HI titres were 5.3 and 3.2, respectively. All commercial and village chickens vaccinated by eye drop survived the challenge trial whereas village chickens vaccinated through drinking water and food had protection levels of 80% and 60% 30 days after primary vaccination, respectively. However, 30 days after booster vaccination, the protection level was 100%. At 60 days after secondary vaccination, the protection level dropped again to 80% for chickens vaccinated orally. All control chickens used in the challenge trials developed clinical ND and died 3-5 days after inoculation with the virulent virus. Supported by laboratory findings, I2 strain of NDV seemed to be avirulent, immunogenic and highly protective against virulent isolates of NDV. It may be a suitable vaccine to use in village chickens to vaccinate them against ND in rural areas.

Rinderpest antibody detected in sheep and goats before an outbreak of rinderpest reported in cattle in northern Tanzania.

In January 1997, serum samples from 1346 adult sheep and goats were tested by a competitive ELISA to determine the prevalence of rinderpest in the northern zone of Tanzania. Seroconversion rates of 20%, 13%, 9%, 7% and 3% in sheep and goats were recorded in Ngorongoro, Monduli, Hai, Arumeru and Simanjiro districts, respectively. The low profile and insidious nature of the rinderpest virus involved caused very mild disease in cattle in some of these area. The mild signs associated with this outbreak of rinderpest resulted in difficulty in its diagnosis. In these circumstances, the presence of rinderpest antibody in sheep and goats served as a valuable and effective indicator of the rinderpest outbreak in cattle.

Breed-associated resistance to tick infestation in Bos indicus and their crosses with Bos taurus.

The relative resistance to tick infestation of zebu (Bos indicus) in comparison to crossbred (B. indicus x B. taurus) cattle was investigated. B. indicus breeds, all belonging to Tanganyika shorthorn zebu were Meru, Mbullu and Iringa red. Crossbreds were Meru x Friesian and Iringa red x Friesian. Parameters to distinguish between 'tick resistant' and 'tick susceptible' cattle were tick counts on naturally exposed animals, serum complement levels and delayed skin hypersensitivity response to phytohaemagglutinin. Results have shown that pure zebu cattle are less infested with ticks when compared to zebu-taurine crosses under identical field conditions. Zebu cattle also had significantly higher serum complement level than crossbred cattle. While serum complement and tick burden were negatively associated (r = -0.27, P < 0.001), the cutaneous response to phytohaemagglutinin did not vary with tick infestation. The influence of cattle breed on tick infestation and serum complement level is demonstrated.