Today was sadly our last day of externship. We woke up early to conduct a milk test in a dairy farm in Sitio Lomboy. We went with Doc Chat, Doc Jonathan and Doc Duran and two other vet students from CLSU. It was very early and it was cold when we arrived at the farm. The farm staff already prepared the buckets and the portable milking machines we will be using. Doc Jonathan prepared the 50 ml vials where we will be placing some milk samples, the CMT reagents and the CMT paddles.
The water buffaloes were guided to a double-sided herringbone milking parlor with one side only used for 19 buffaloes at a time. Feeds were placed in the feeders and the buffaloes which were known to be aggressive were tied to the post at the hindlimb. One of the farm staff then started forestripping the teats of each animal after he wiped them with a clean cloth. Labels using tapes were placed on each division to denote each animal for easier identification. We then conducted the CMT while the farm staff acquire milk samples using the paddles. Doc Jonathan was recording our observations.
Lastly, before the animals are led out of the parlor, bulk milk was collected using the portable milking machine.
The teats were dipped on povidone iodine solution after milking to prevent mastitis. The bulk milk was weighed and recorded in kilograms. Bulk milk negative for the CMT was then transferred to a collecting bucket after straining in a cloth and after being sampled in a vial.
The sun was already shining at us when we finished testing and sampling all the animals.
We went back to the PCC headquarters to be able to submit the milk samples at the Milk and Meat Laboratory for analysis.
In the afternoon, we went back to the same dairy farm with
some new vials containing Bronopol, a preservative and an ice box. We arrived
at the farm at just the right time for the afternoon milking. We collected
negative bulk milk samples, placed them in vials and transported them in an ice
box. The milk collected was again for milk analysis.
Today was supposed to be a Milk Test day but the cooperative at Riverside had a power and water shortage and so Doc Chat decided to postpone the testing and so we headed back to the laboratory.
At the laboratory, we helped sir Dadz extract DNA from blood samples and Mam Noemi in doing PCR.
We also observed in making agar gel for electrophoreses.
In the afternoon, we helped Mam Noemi do some subcultures of a bacterial culture and sir Dadz with RBPT.
We started the day by processing fecal samples and observing them under the microscope.
Afterwards, we also extracted DNA from some urine samples.
Some of us were assigned to check the blood samples for microfilaria or Trypanosoma spp. The blood samples were placed in capillary tubes and centrifuged for 5 minutes at 10000 rpm. The buffy coat were observed for the blood parasites under the microscope. The PCV was also acquired and then recorded. The blood samples for those tests were from the purple top tubes containing EDTA.
The
blood samples in the plain vials were used for the RBPT. 25 ul of the serum
from those blood samples were mixed with 25 ul of reagent for the Brucella spp. testing.
After observing some coccidia under the microscope during fecalysis, Sir Dadz told us to extract its DNA. We did so by doing the following steps:
Add 0.5 ml of fecal sample in a 1.5 MCT.
Add glass beads until the 1 ml mark.
Add 0.5 ml of lysing solution.
Vortex the mixture for 30 seconds and then
incubate at 95 degrees Celsius for 15 minutes.
Centrifuge at 14 000 rpm for 1 minute. Aspirate
the supernatant and transfer into a 1.5 MCT with 500 ul of isopropanol.
Centrifuge at 14 000 rpm for 1 minute and then
discard the supernatant.
Add 500 ul of 70% ethanol and then discard the
supernatant.
Add 30 ul of DNA rehydration solution.
Incubate at 95 degrees Celsius for 10 minutes
and then store at 20 degrees Celsius until PCR.
For today, some of the PCC staff is still attending their weekly Monday meeting. So, sir Dadz allowed us to go the National Water Buffalo Gene Pool to meet Doc Jim. We rode our bicycles to the Gene Pool and were met by the biosecurity measures such as the tire bath and the fencing. Doc Jim and the vet students from Central Luzon State University were transferring caracows from pen to another. We also saw Tata Mar and Tata Naro conducting pregnancy diagnosis through rectal palpation just like they do whenever we go out to the National Impact Zone or to other dairy farms.
We went around to see the pens and saw some cattle mixed with the water buffaloes.
It was also feeding time for the caracalves and we saw one albino water buffalo which was a progeny of some albino lineage in the farm.
On our way back to the laboratory, we passed by a small building with pens for goats specifically Boer goats which are meat-type of animals.
Back at the laboratory, we helped Sir Dadz in processing some fecal samples and observed a lot of Fasciola spp. eggs in the samples after sedimentation technique.
Some urine samples also arrived at the laboratory and Sir
Dadz instructed us to get the DNA samples. The method was as follows:
Transfer 1 ml of urine into a 2 ml tube.
Centrifuge 14000 rpm at 10 degrees Celsius for
10 minutes.
Today, we attended a proposal of a product by Mr. Aner Yacobi entitled SCR Fertility and Cow Welfare. He proposes to PCC staff, veterinarians and heads of offices an electronic ear tag, yet to be released in the country, which measures the activity of a cow to detect estrus as well as to measure the rumination time of a cow which correlates to its health and nutrition. Mr. Yacobi said that cud chewing is a factor where nutritionists in their country heavily rely on to assess the nutrition of the animal. One of their products already released is a collar type which measures movement of the neck. However, the only problem they encounter with this design is strangulation of the animal.
Mr. Yacobi started by saying that “If you can’t measure it, you can’t manage it.”. He is referring to the measurement and detection of estrus to allow rebreeding and make an efficient breeding program. He says that the product helps optimize herd health, fertility and nutrition which are keys for profitability. The product sends data to a modem and then sends the data to a computer which can be placed in an office. Data can easily be managed and read since they are immediately presented in tables and graphs. However, training is required before interpretations can be done. The product has never been tested to buffaloes yet. PCC is willing to conduct trials to detect the accuracy of such products to their own herd.
Just before lunch, we taught Novy how to extract DNA from some blood samples. After lunch, we went to observe Mam Noemi who was examining three disposable plastic dishes containing a fungal culture she made from paddy straws. She aims to isolate the causative agent for Degnala disease which is Fusarium spp.. She also made an enriched culture. It was quite difficult to acquire fungi from the culture and place them on glass slides for observation under the microscope unlike for bacteria. We then decided to use Scotch tape test to acquire some fungi from the plates. We stuck to tapes to the glass slides and slightly stained them with methylene blue. Based on our examinations, the cultures contain not Fusarium spp. but Aspergillus spp. based on morphology. Aspergillus spp. is a ubiquitious fungi and is an opportunistic pathogen. We disinfected immediately the slides and materials we used and then went on to process the purulent fluid we collected yesterday.
With the help of Mam Noemi, we inoculated some of the purulent fluid into three plates containing nutrient agar (NA). NA is a general purpose medium which supports the growth of a wide range of non-fastidious organisms. We then stained some of the purulent fluid on a glass slide using Gram stain (1 minute crystal violet, 1 minute Lugol’s iodine, 10 seconds alcohol and 30 seconds safranin). We observed some gram positive cocci in few numbers and suspected that they were just contaminants. We will wait for the culture results and then stain again later to observe for the bacteria which will grow.
Today was another Milk Test Day and it was Novy’s first time to attend the activity so we guided her aside from Doc Chat’s tutorial. We left the headquarters at 6 in the morning together with Doc Bjet and Doc Jonathan. We arrived then in one of the cooperatives supported by PCC, Pulong Buli Primary Multi-Purpose Coperative in Sto. Domingo, Nueva Ecija. We immediately started with the milk testing using the California Mastitis Test. All the negative samples for CMT were placed in milk cans to be pasteurized and sold as dairy products. Some amount of the negative samples was placed in 50 ml tubes to be submitted at the Meat and Milk laboratory to be analyzed for contents. Mam Paulen said last week that those cows yielding the best quality milk based on the milk analyzer will be used for breeding to improve the production of each dairy farm and in other words, the cooperative itself.
There was a meeting after the milk testing and Doc Bjet asked who were interested to avail carabull or caracow loans from PCC. After the meeting, we accompanied Doc Bjet in going to Saranay or LBRAF where the buffaloes for loan are being housed. Doc Bjet who was a roaming veterinarian in PCC was also responsible for checking some of the buffaloes for loan and transacting them to those interested to avail.
In the afternoon, we headed back to Sto. Domingo because a farmer called Doc Bjet for a case in his farm. According to the farmer, his caracalf has a swelling in the abdomen and Doc Bjet suspected it was umbilical hernia. He instructed us to prepare for a surgery and so we wore our boots and gloves, carried the surgical instruments, drapes, drugs and other paraphernalia to the pen after it was cleaned by the farmer.
Doc Bjet immediately saw the swelling since it was already big, the size of a medium melon. Doc Bjet palpated the swelling and it was soft and feels like a fluid inside is present. Doc Bjet suspected it was not umbilical hernia but navel ill or omphalitis. It is a condition wherein the navel or umbilicus of a caracalf is infected by contamination of bacteria which is usually found in the environment. This condition is common usually soon after birth and is localized. However, Doc Bjet said that such cases should be attended immediately since the swelling may serve as a source of infection leading to septicemia. The caracalf was 1 month old and is still active and not feverish which indicates a good prognosis.
With the help of the farm owners, the calf was restrained by tying its neck to a fence and tying both the front legs together as well as the hind legs. When the caracalf was already in lateral recumbency, one of the owners was holding it down at the head region while some of us were restraining it near the tail region. The umbilicus can better be seen and it was enlarged, somewhat painful upon palpation but is not draining any purulent material through a fistula. Doc Bjet inserted an 18-gauge needle connected to a 50 ml syringe through the swelling and began aspirating. Doc Bjet aspirated milky white, purulent fluid from the swelling and confirmed that it is omphalitis based on his experience in the field.
Doc Bjet decided to open the swelling to drain the pus faster and to clean the inside of the swelling. Previously prepared povidone iodine solution was sprayed onto the surface of the swelling and the swelling was shaved with a blade to remove the hairs. Using a different blade, a #20 scalpel blade, a small incision was made on the swelling and pus was drained to a steel basin. Approximately 600 ml of pus was drained by continuously massaging the swelling. When a maximum amount of pus was already drained through massage, the inside of the swelling was flushed with povidone iodine solution using a syringe with an attached sterile cut AI catheter to reach further inside. This technique is common and we’ve learned this during our small ruminant duty where povidone iodine can be used for uterine lavage to clean the uterus of bacteria. Povidone iodine is an effective antiseptic and is effective against a wide range of bacteria. Therapeutic irrigation or lavage was done several times until no pus can be observed going out with the solution.
The inside was then sprayed with chlortetracycline hydrochloride, a tetracycline antibacterial effective against many opportunistic bacteria both gram-positive (Staphylococcus spp. and Streptococcus spp.) and gram-negative (Bacillus spp., Clostridium spp., etc.). Next, a screw worm aerosol was used aas a wound spray to prevent myiasis or the infestation of the wound opening with fly larvae. Its active ingredient is permethrin which is an insecticide that kills flies and maggots that come in contact with it. Intramuscular injection with an antibiotic, amoxicillin + gentamicin, was also done since topical antibiotics are not that effective for treatment alone and in this case was only a supplement to intravenous antibiotics. Tolfenamic acid was also administered intramuscularly in a site different from the first intramuscular injection as an anti-inflammatory drug and analgesic. Lastly, tetanus toxoid or tetanus vaccine, an inactive vaccine used to prevent tetanus, was injected at the gluteal muscles. Doc Bjet said that tetanus toxoid must always be given when doing surgery especially when wounds are being created because tetanus (caused by the bacteria Clostridium tetani) is common and can be deadly. Doc Bjet said that he will go back to the farm to administer antibiotics for the following days and to check the prognosis of the condition. We brought some of the purulent fluid back to the laboratory and stored it for culturing and morphological staining.
For today, we were assigned to Tata Mar together with Novy, a new veterinary student intern from NVSU. We accompanied Tata Mar in going to dairy farms and extending the services of PCC such as deworming, vitamin administration and pregnancy diagnosis. We guided Novy in our usual routine especially in drug administration as well as in record keeping. Tata Mar taught her rectal palpation.
We also dewormed all the caracalves in one dairy farm with a new drug we encountered in the field which is doramectin. Just like ivermectin which we were accustomed to using, this drug is also an avermectin antiparasitic agent. Based on its label, the drug is usually indicated for use as an injectable in cattle and swine and as a topical in cattle. It is indicated for the treatment and contol of the following endo- and ectoparasites in cattle: roundworms (adults and some fourth stage larvae)—Ostertagia ostertagi (including inhibited larvae), O. lyrata, Haemonchus placei, Trichostrongylus axei,T. colubriformis,T. longispicularis, Cooperia oncophora, C. pectinata,C. punctata, C. surnabada (syn. mcmasteri), Bunostomum phlebotomum,Strongyloides papillosus, Oesophagostomum radiatum,Trichuris spp.; lungworms (adults and fourth stage larvae)—Dictyocaulusviviparus; eyeworms (adults)—Thelazia spp.; grubs (parasitic stages)—Hypoderma bovis, H. lineatum; lice—Haematopinuseurysternus, Linognathus vituli, Solenopotes capillatus; and mange mites—Psoroptes bovis, Sarcoptes scabiei. The manufacturer also states that doramectin protects cattle against infection or reinfection with Ostertagia ostertagi for up to 21 days.
While we were about to leave and go to another farm, we noticed a tricyle driver delivering some grasses to feed to the buffaloes. The grass was not the usual Napier grass we see that were being fed to buffaloes. The grass was barnyard or cockspur grass (Echinochloa crus-galli), a type of wild grass in the country which was previously classified as a type of panicum grass. It was one of the worst weeds and can be found along roadsides, ditches, along railway lines, in disturbed areas such as gravel pits and dumps, riverbanks and the shores of lakes and ponds. It was removed by the farm boys who delivered the grasses since it was a nuisance and for buffalo feed as well. These warm-seaspm grasses were documented to have been used in some countries as cattle fodder throughout the year and is sometimes cultivated for that purpose. Some authors said it was suited for silage but not for hay. Tata Mar said that while it can be fed, it provides lower quality of fiber and nutrients as compared to Napier.
The next farm we went to was at the top of a hill overlooking onion farmlands and it was very windy and has a breathtaking view. This farm is also an integrated farm with swine pens and free range poultry. The chickens were also beneficiaries of the leftover feeds from carabao feed according to the farm manager which is an advantage since nothing or minimal feed is wasted. We also noticed hay which were compacted, bundled or baled. They used a large round hay baler, a farm machinery used to compress and roll cut and raked hay. This method was done for the ease of handling, transport and storage of hay. The cylindrical hay was also bound by a wire which Tata Mar said to dispose of properly since the buffaloes may accidentally ingest them.
After
the caracows underwent pregnancy diagnoses through rectal palpation, we noticed
one caracow which has horns curling back to its head and nearly puncturing it. Tata
Mar then asked for a steel saw and began to saw the distal 1/5 of the horn. After
sawing the tips of the horns, Tata Mar also asked for a whetstone to remove the
sharp edges of the horn and prevent future damages. The procedure was quite
fast and easy and did not require prior anesthetic administration and
post-operative wound management since there was no bleeding.
Sir Dadz told us that we still don’t have a scheduled assignment for today since most of the samples have already been tested (i.e. fecalysis and RBPT) and processed (i.e. DNA extraction) last week. We decided to just review some of our old notes for the mean time. Mam Jona from the Meat and Milk laboratory of PCC texted Sir Dadz and telling him to get some blood samples from their laboratory. James and I went to the laboratory and she handed us an ice box containing some blood samples in plain vials. Based on the labels, these were from the Ubay Stock Farm of PCC in Bohol.
Also called Ubay Agri-Park, is one of the oldest and also the largest government livestock facility in the country. Based on the stories of some of the PCC staff, the facility also houses different farm animals aside from the carabao which are cows, horses, goats and poultry such as chickens, turkeys, and ducks. Following one of the core principles of PCC, the facility also helps in developing the livelihood of farmers while also catering to tourists who would like to have some fresh milk and other dairy products.
The facility
has sent some samples for screening of some diseases. So for the whole day, all
of us extracted the DNA from all 105 samples. We followed our usual protocol
for DNA extraction:
In
a 1.5 ml microcentrifuge tube (MCT), 1000ul of NH4Cl were added to 500ul of
buffy coat/whole blood sample.
The
mixture was mixed by pipetting/vortex mixer, and then centrifuged at 14000 rpm
for 1 minute.
The
supernatant was discarded.
White
pellets after centrifugation were observed. Then, steps 1-3 were repeated.
1000
ul of Cell lysis was then added, the mixture was vortexed to dislodge the
pellet, and then it was centrifuged at 14000 rpm for 1 minute.
The
supernatant was then discarded by pipetting.
300
ul of Nuclei lysis solution was added.
The
mixture was again vortexed for at least 30-40 seconds.
100
ul of Protein precipitate was added, and then the mixture was vortexed until it
was homogenized.
Centrifugation
was also done at 14000 rpm for 10 minutes.
While waiting, new 1.5 MCT were prepared with
each containing 500 ul of isopropanol/propanol.
The
clear liquid after centrifugation of samples was transferred to the new 1.5 MCT
and then mixed with inversion.
The
mixture was then centrifuged at 14000 rpm for 1 minute.
The
supernatant was discarded.
500
ul of 70 % Ethyl alcohol was added.
The
mixture was then centrifuged at 14 000 rpm for 1 minute.
The
supernatant was then discarded.
The
tubes were air dried for at least 30-40 minutes without overdrying the DNA.
Rehydration
was then done by adding 30 ul of DNA rehydration solution.
The DNA samples were incubated at 65 degrees Celsius for 10 minutes and then stored in 4 degrees Celsius.
When we entered the Biosafety and Environment laboratory, we went straight to the incubator and observed the culture media we inoculated yesterday. We observed what we expected that was that the last line of streaking had the thinnest lines and many isolated colonies of bacteria can be observed. These colonies can be further described by using some specific terminologies. First is the form or the basic shape of the colony such as circular, filamentous, etc. The size or the diameter of the colonies can also be measured using a ruler. Some also measure the side view of the colonies or their elevation viewed when the petri dish is turned on one end. On a closer look, the margin or the border of the colony can be magnified and observed as well as for its surface (smooth, glistening, rough, wrinkled or dull). Other colony characteristics that can be observed are its opacity (transparent or clear, opaque, translucent, etc. ) and the pigmentation or color (white, red, purple, etc.).The colonies we observed were similar in overall morphology which means that it is possible that they are of the same group. When colonies are distinct, each colony will represent an individual bacterial cell group.
We then proceeded to the microbiology room and made stock cultures of the colonies in our culture media. These will be kept so that the microorganisms will be in viable condition or so that there will still be a source of the microorganisms if they will be needed. We observed aseptic techniques such as flaming the loops and then dubbing it on to a bacterial colony. The inoculationg loop or needle is then stabbed on to a small tube containing a culture media and then they will be stored in the refrigerator. Also, some colonies were placed using inoculating needles in small microcentrifuge tubes containing double distilled water. They were then placed in a 95 degrees Celsius water bath and this is the Boiling Method of bacterial DNA extraction.
While waiting for the microcentrifuge tubes with bacterial colonies, we helped in testing some blood samples sent to the laboratory for Brucella spp. using the RBPT.
For each of the organ sample’s (lungs, liver, mesenteric lymph node) culture media, 2 representative colonies were taken so there were 6 total samples. After boiling them to extract their DNA, they were then used for PCR mixture preparation. The master mix consists of 56.8 ul of double distilled water, 20 ul of buffer, 40 ul MgCl2, 24 ul dNTP, 8 ul each of forward and reverse primers and 3.2 ul of Taq polymerase. Five microliters of DNA for each sample was then mixed with a corresponding amount of the mixture.
The PCR tubes containing the PCR mixture were then placed inside the thermal cycler or the PCR machine and the run time was approximately 2 hours. Thermal cyclers are “DNA amplifiers” which regulate the temperature in different cycles (denaturation, annealing and extension). The following steps will not be done yet. Mam Noemi said that the PCR products will still be further processed, will be sequenced through an automated DNA sequencer and then the sequence results will be analysed by the BLAST search engine.
Huwaw! Karabaw 